Protein Sample Ultrafiltration Protocols

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Most Amicon® users know that ultrafiltration is an ideal method for concentrating, desalting, and exchanging buffers of protein samples. But did you know that you can use ultrafiltration devices for other applications, such as cleaning up labeling reactions, purifying antibodies, and removing detergents?

Maybe you’ve seen such methods cited in a publication, but the “Materials and Methods” section you read just isn’t detailed enough.

On this page, you will find links to step-by-step protocols for using ultrafiltration membrane-based devices for these applications and others.

Protocols for Buffer Exchange and Concentration:

Click on links to view protocols. 

Concentration, Desalting, and Buffer Exchange with Amicon® Ultra Centrifugal Filters

Amicon® Ultra centrifugal filters are ideal for removal or exchange of salts, sugars, nucleotides, and non-aqueous solvents, as well as other materials of low molecular weight. They also serve to separate free and bound species. One of the most common applications for Amicon® devices is concentration and desalting of column fractions during protein purification by various chromatography methods.

Use this protocol for:
  • Dilute or purified protein solutes, antigens, antibodies, enzymes, or microorganisms
  • Fast, convenient, high-recovery alternatives to dialysis and ethanol precipitation
  • Efficient salt removal independent of biomolecule concentration or size

Amicon® Ultra Method for Concentration, Desalting or Buffer Exchange

  1. Using the Amicon® Ultra Online Selector Tool, select the Amicon® Ultra device with the appropriate nominal molecular weight limit (NMWL) and volume capacity for your application.
  2. (Optional) Pre-rinse device with buffer of choice.
  3. Add the sample to the reservoir of the centrifugal device.
  4. If the sample is smaller than the maximum volume, it can be diluted up to the maximum volume before the first centrifugation step. This will help increase salt removal.
  5. Centrifuge at the specified g-force for the amount of time recommended in the user guide for the selected device [click to browse user guides].
  6. Remove the initial filtrate from the filtrate tube and set aside.
  7. Add enough buffer (for buffer exchange) or water (for desalting) to the device to bring the sample volume up to 4, 15, 2, or 0.5 mL, depending on the volume capacity of the device.
  8. Centrifuge again.
  9. Set aside the filtrate.
  10. Recover the concentrated, desalted, or buffer-exchanged sample.
NOTE: Both of the filtrates should be retained until the concentrated sample has been analyzed.

Principles

During the first centrifugation step, the concentration of macromolecule solute increases; however, filtration removes lower MW solutes, thereby maintaining the salt concentration at 500 mM. For the second spin, another volume of salt-free buffer or water is passed over the retentate, and salt is removed by filtration. This process of “diafiltration” can be repeated to achieve maximum salt removal, or complete buffer exchange.

Amicon® Ultra centrifugal devices allow >90% salt removal in the first centrifugation step. As seen in the table below, one additional centrifugation step generally increases the salt removal to 99% with >90% recovery of the sample.

Cytochrome c (0.25 mg/mL)
BSA 1 mg/mL
BSA 1 mg/mL
IgG 1 mg/mL
Device
Amicon® Ultra 15 mL filter with 10 kDa NMWL Amicon® Ultra 15 mL filter with 30 kDa NMWL Amicon® Ultra 15 mL filter with 50 kDa NMWL Amicon® Ultra 15 mL filter with 100 kDa NMWL
Spin
1 2 1 2 1 2 1 2
% Protein Recovery
97.3 95.6 96.0 94.4 98.9 92.4 99.9 97.1
% NaCl Removal
97.9 99.9 98.2 99.9 97.1 99.9 97.7 99.5

Table: Removal of sodium chloride and recovery of protein with Amicon® Ultra-15 mL devices.

Three Amicon® Ultra-15 devices of each NMWL indicated were tested with 15 mL of each protein solution containing 500 mM initial concentration of NaCl. Each spin was performed at 4,000 x g for 30 minutes. After the first spin, the retentate was brought up to 15 mL with ultrapure water from a Milli-Q® water system. NaCl removal was calculated by assaying each sample for conductivity (µS/cm) using a standard conductivity meter (Innolab® Conductivity Meter). Protein recovery was calculated by measuring the final amount of protein in the retentate after each spin and comparing to the amount of protein loaded. Amount of protein in the retentate was calculated by measuring the absorbance of the retentate solution at 410 nm (for Cytochrome c) and at 280 nm (for BSA and IgG) and recovery calculated using the following equation:

Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/psp-protocols-equation.jpg

Where ...

W= total weight of retentate before assay

W= weight of original starting material

C= concentration of protein in retentate

C= original starting material concentration


NOTE: Factors independent of the ultrafiltration device may contribute to low protein recovery. Two examples:
  • Desalting below a certain threshold for a given protein can result in protein precipitation and lower measured protein recovery.
  • Overconcentration of protein can result in lower measured protein recovery.
For single-spin buffer exchange of small samples and with simultaneous protein concentration, refer to the Amicon® Pro system protocol for continuous diafiltration and concentration.


Buffer Exchange and Concentration Using Amicon® Pro Centrifugal Filters for Continuous Diafiltration

Even more efficient desalting with simultaneous macrosolute concentration can be achieved using the Amicon® Pro device, which was specifically engineered for effective buffer exchange (≥ 99%) with a single spin (see figure below). Furthermore, the Amicon® Pro device enables continuous diafiltration. Continuous diafiltration maintains a constant volume throughout the buffer exchange process, by matching the rate at which exchange buffer is introduced to the filtrate flow. Because the volume remains constant, the concentration of retained solute also remains constant, providing a gentler method of buffer exchange.

As shown in another protocol, buffer exchange is thus completed in a single step, instead of three steps as required for traditional diafiltration methods (see data comparing traditional and continuous diafiltration).

Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/amicon-pro-device-single-spin.jpg
Amicon® Pro device for single-spin desalting and buffer exchange. Specific design components include: (1) large upper reservoir of the exchange device, (2) tight contour matching of the exchange device to the Amicon® Ultra 0.5 mL filter to minimize internal spacing when the two devices are connected, and (3) tapered exchange device tip for optimal buffering.

Amicon® Pro Method for Desalting and Concentration:

  1. Select the Amicon® Pro device with the appropriate nominal molecular weight limit (NMWL) for your application.
  2. Optional: Pre-wet device using the particular buffer solution you are using, for example, 0.5 mL TBS-T. Spin 1000 x g for 1 min. In cases of highly dilute samples, a passivation protocol with detergent may be beneficial. In cases of mass spectrometry applications, use mass spectrometry-compatible detergent (such as deoxycholate).
  3. Optional Wash: Add 1.5 mL appropriate buffer. Spin 1000 x g for 1 min.
  4. Attach the included Amicon® Ultra 0.5 mL filter (five MWCO options available) to the exchange device.
  5. Add 1.5 mL* of sample to the exchange device Spin 4000 x g for 15 min to concentrate.
  6. Add 1.5 mL* of desired buffer. Spin 4000 x g for 15 min to exchange buffer and concentrate.
  7. Recover purified protein from the Amicon® Ultra 0.5 filter by reverse spin.
*For larger sample/buffer volumes, spin time will need to be increased appropriately. Please consult user guide for specific instructions.

NOTE: Do not load more than 2 mg total protein in step 5. Exceeding 2 mg will result in protein precipitation upon concentration.

Results

With continuous diafiltration, as occurs in the Amicon® Pro device, the sample (after it has undergone initial concentration and has entered the Amicon® Ultra-0.5 mL component of the Amicon® Pro system) remains at a constant volume (and concentration) until the entire quantity loaded in the upper reservoir has been passed through the exchange device tip. In this experiment, over 99.9% of the salt was removed after a single spin (See table below). The Amicon® Pro device also afforded extremely high protein recovery, having, on average, < 2% loss of starting BSA.  

BSA 0.05 mg/mL
Device
Amicon® Pro device (with Amicon® Ultra 10 kDa NMWL filter attached)
Spin
% Protein Recovery % NaCl Removal
1
Not determined (initial protein concentration step) 0% (NaCl concentration unchanged during proteinconcentration step)
2
>98% >99.9%

Table: Removal of sodium chloride and recovery of protein with Amicon® Pro device.

500 µL of a BSA solution was subjected to simultaneous desalting (using 1 M Tris, pH 7.5) and concentration. The resulting sample was brought to an equivalent volume in 1 M Tris pH 7.5, diluted 1:50 in Milli-Q® H20, and assayed for conductivity (µS/cm) using a standard conductivity meter (InoLab® Cond). Protein concentration was determined using the Direct Detect® Infrared Spectrometer for Protein Quantitation.


Detergent Removal with Amicon® Pro Centrifugal Filters

Detergents are frequently used to solubilize proteins and nucleic acids during purification, but the presence of detergents may interfere with downstream analyses. Free detergent can be removed by various protocols1; however, detergent molecules that are noncovalently bound to macromolecules can be displaced but not completely removed. Fortunately, for most downstream analyses, it is sufficient to remove the free detergent.

Amicon® Pro centrifugal filters are efficient tools for removing detergents from solutions of proteins or nucleic acids. The choice of filter depends on the critical micelle concentration (CMC) for a given detergent. At concentrations above the CMC, detergent monomers form micelle aggregates with gross changes in molecular structure. Choosing a centrifugal device with the correct nominal molecular weight limit (NMWL) allows for effective detergent removal.

However, it may be challenging to choose an ultrafiltration device with a NMWL that allows detergents, but not protein of interest, to pass through. Because they form large micelles, many detergents require using membranes with NMWL as high as 100 kDa for size-based depletion, resulting in loss of the proteins of interest in the sample. One way to use smaller pore size ultrafilters for these detergents is to reduce micelle formation first, by sample dilution.

The Amicon® Pro system was used to remove detergents using continuous diafiltration, in which a constant sample volume is continously washed with detergent-free buffer. This method could remove detergents more efficiently and with higher protein recovery than using discontinuous diafiltration or dialysis. Simultaneous monitoring detergent removal and protein recovery was performed using the Direct Detect® infrared spectrometer.

For more information on detergent removal monitoring methods, please refer to our publication, Detergent Analysis in Protein Samples Using Mid-Infrared (MIR) Spectroscopy.

Method

  1. If necessary, clarify the sample to be analyzed using a Steriflip® 0.45 µm filter or a Millex® 0.45 µm syringe filter.
  2. Use the Direct Detect® spectrometer to estimate concentrations of detergent and protein in the starting material. Refer to the Direct Detect® User Guide, as well as our publication, for details on calibrating the spectrometer using an 8-point dilution of the detergent of interest and the instrument software’s “lipid calibration wizard”.
  3. Add 50-100 µL of this sample to the Amicon® Ultra 0.5 mL filter (included in the Amicon® Pro system)
  4. Attach the filter to the Amicon® Pro exchange device.
  5. Add 1 mL of phosphate-buffered saline to the exchange device/Amicon® Ultra 0.5 mL filter assembly. Fully assemble Amicon® Pro device.

    (The Amicon® Pro device can safely accommodate up to 9 mL of buffer. However, adding just 1.0 mL of fresh buffer will effectively enable 1000-fold buffer exchange, because of the efficiency of continous diafiltration in constantly exposing the sample to fresh, detergent-free buffer.)
     
  6. Centrifuge at 4,000 x g for 30 minutes in a swinging-bucket rotor.
  7. Remove and disassemble the Amicon® Pro device.
  8. Place the Amicon® Ultra 0.5 mL collection tube over the top of the Amicon® Ultra 0.5 mL filter and invert.
  9. Recover the sample by spinning in a fixed-angle rotor at 1,000 × g for 2 minutes.
  10. Use the Direct Detect® spectrometer as in step 2 to estimate concentration of recovered protein and to confirm detergent displacement.

Example Results of Detergent Removal Using the Amicon® Pro System

Removal of 0.5% sodium deoxycholate from 2 mg/mL IgG. Using 2 mg/mL IgG solubilized in 0.5% sodium deoxycholate, the Amicon® Pro purification system was used to show that detergent diluted below its CMC could be easily removed by a size-based ultrafiltration method.

The figure below compares detergent removal via continuous diafiltration with successive dilution and concentration steps. Continuous diafiltration keeps the concentration of free detergent below the critical micelle concentration (CMC). As a result, fewer micelles form than with alternating rounds of dilution and concentration, and ultrafiltration is more effective at removing detergent molecules.

Continuous diafiltration also avoids concentrating protein too rapidly in a detergent-free buffer, a process which may disrupt stabilizing detergent-protein interactions, cause protein precipitation, and reduce yield.

Measuring detergent removal and protein recovery during detergent removal made it possible to identify the protocol that yielded the optimal balance of both processes.

Detergent was removed from a 50 µL protein sample while monitoring percent detergent removed (A) as well as percent protein recovered (B). Gray bars reflect the results of removing detergent using continuous diafiltration using 10x, 20x and 30x volumes of detergent-free buffer, while the black bars reflect the results of removing detergent by first diluting the sample by 10x, 20x, or 30x, followed by concentration by ultrafiltration.   Reference: 1.  Das C, Nadler T, Strug I. Detergent Analysis in Protein Samples Using Mid-Infrared (MIR) Spectroscopy. Curr Protoc Protein Sci. 2015 Aug 3;81:29.12.1-15.  Concentrating Large Volumes With Centricon® Plus-70 Centrifugal Filter Device  Large-volume centrifugal filter devices are a convenient alternative to stirred cell ultrafiltration devices for purifying proteins from large volumes of solution. Stirred cells are gentle, efficient, and feature a removable membrane for greatest flexibility. However, centrifugal ultrafilters have their own advantages: Devices are pre-assembled and ready to use Can process 20 or 70 mL volumes; use multiple devices for larger volumes  Spin times are fast (minutes) No need to refill the reservoir (less contamination risk) The following protocol is an example that demonstrates the use of Centricon® Plus-70 centrifugal filter devices to purify and concentrate a fusion protein composed of the alpha and gamma subunits of the human high affinity IgE receptor. The alpha-gamma fusion protein is purified and then coupled to Sepharose® (GE) beads to produce an affinity column for isolating human IgE antibodies. (Note: protocol courtesy of GKT School of Biomedical Sciences, London.) Method The alpha and gamma subunit components of the high affinity IgE receptor were transfected into mouse myeloma cell line NS0 and the secreted fusion protein was purified on a Protein G column. The eluted proteins were concentrated from volumes of 500–1000 mL to 5–10 mL using a Centricon® Plus-70 centrifugal filter device in a swinging bucket rotor at 3000 x g for 10 minutes (60 mL per filter unit). Buffer exchange was also performed using the same units in tissue culture grade sodium azide-free PBS. The concentrated samples were filter-sterilized under sterile conditions using a 0.2 μm pore filter. Results According to spectrophotometric analysis, 18.8 mg/L of alpha-gamma fusion protein was recovered using the Centricon® Plus-70 devices (data not shown). The HPLC trace analysis revealed the profile of the alpha gamma fusion component of the human FcεRI (See figure below).
Detergent was removed from a 50 µL protein sample while monitoring percent detergent removed (A) as well as percent protein recovered (B). Gray bars reflect the results of removing detergent using continuous diafiltration using 10x, 20x and 30x volumes of detergent-free buffer, while the black bars reflect the results of removing detergent by first diluting the sample by 10x, 20x, or 30x, followed by concentration by ultrafiltration. Note that when the sample was only diluted 10-fold, traditional ultrafiltration concentration did not remove any detergent from the sample (A).
 
Reference:
1.  Das C, Nadler T, Strug I. Detergent Analysis in Protein Samples Using Mid-Infrared (MIR) Spectroscopy. Curr Protoc Protein Sci. 2015 Aug 3;81:29.12.1-15.




Concentrating Large Volumes With Centricon® Plus-70 Centrifugal Filter Device

Large-volume centrifugal filter devices are a convenient alternative to stirred cell ultrafiltration devices for purifying proteins from large volumes of solution. Stirred cells are gentle, efficient, and feature a removable membrane for greatest flexibility. However, centrifugal ultrafilters have their own advantages:
  • Devices are pre-assembled and ready to use
  • Can process 15 – 65 mL volumes; use multiple devices for larger volumes 
  • Spin times are fast (minutes)
  • No need to refill the reservoir (less contamination risk)
The following protocol is an example that demonstrates the use of Centricon® Plus-70 centrifugal filter devices to purify and concentrate a fusion protein composed of the alpha and gamma subunits of the human high affinity IgE receptor. The alpha-gamma fusion protein is purified and then coupled to Sepharose® (GE) beads to produce an affinity column for isolating human IgE antibodies. (Note: protocol courtesy of GKT School of Biomedical Sciences, London.)

Method

  1. The alpha and gamma subunit components of the high affinity IgE receptor were transfected into mouse myeloma cell line NS0 and the secreted fusion protein was purified on a Protein G column.
  2. The eluted proteins were concentrated from volumes of 500–1000 mL to 5–10 mL using a Centricon® Plus-70 centrifugal filter device in a swinging bucket rotor at 3000 x g for 10 minutes (60 mL per filter unit).
  3. Buffer exchange was also performed using the same units in tissue culture grade sodium azide-free PBS.
  4. The concentrated samples were filter-sterilized under sterile conditions using a 0.2 μm pore filter.

Results

According to spectrophotometric analysis, 18.8 mg/L of alpha-gamma fusion protein was recovered using the Centricon® Plus-70 devices (data not shown). The HPLC trace analysis revealed the profile of the alpha gamma fusion component of the human FcεRI (See figure below).

HPLC profile of alpha-gamma fusion component of human FcɛRI
HPLC profile of alpha-gamma fusion component of human FcɛRI

Protocols for General Affinity Purification:

Click on links to view protocols. 

Affinity Purification with Ultrafree®-MC Centrifugal Filters

Affinity interaction chromatography is an effective method for protein purification that can achieve up to 95% purity in one step. It is often employed in the small-scale batch mode as a quick method for microgram-scale protein purification. The typical protocol involves:
  1. Pipetting a small volume of affinity resin into the microfuge tube containing the sample
  2. Vortexing the tube for a few minutes
  3. Centrifuging the resin to the bottom
  4. Pipetting off the supernatant
  5. Washing a few times (using steps 2 and 3)
  6. Eluting with a small amount of eluent
Pre-packed mini-spin columns (such as Montage® Antibody Purification Kits) for protein purification simplify this process into a one-hour protocol.

For a more flexible protocol, centrifugal devices with a microporous membrane, such as Ultrafree®-MC centrifugal devices, hold the sample and affinity resin in a filter basket, thus creating a “home-made” mini-spin column. These devices combine the high efficiency of batch binding and washing with the handling convenience of a mini-spin column.

With minimal hands-on time, the method provides flexibility in resin-to-lysate ratio and binding conditions, independent of centrifugation speed and rotor angle. Here, the method was used to purify rabbit IgG on ProSep® resin and His-tagged C-RP protein on three different commercial metal-chelate resins.

Method for IgG Purification

Solutions
  • ProSep-A binding buffer A: 1.5 M Glycine/NaOH, 3 M NaCl, pH 9.0
  • ProSep-A elution buffer B2: 0.2 M Glycine/HCl, pH 2.5
  • ProSep-A neutralization buffer: 1 M Tris/HCl, pH 9.0
Procedure
  1. 200 mg of ProSep® resin were placed in Ultrafree®-MC 0.45 μm filter basket.
  2. The columns were equilibrated with 400 μL of binding buffer A and centrifuged for 1 minute at 100 x g.
  3. 200 μL of rabbit serum were diluted 1:1 with binding buffer and the entire volume was loaded into the spin column containing PROSEP-A resin.
  4. Devices were placed on a shaker for 15 minutes at room temperature and centrifuged at 100 x g for 5 minutes. Flow-through was collected for future analysis.
  5. Three consecutive washes were performed, each time adding 400 μL of binding buffer A and centrifuging at 2,000 x g for 2 minutes.
  6. 200 μL of elution buffer B2 were added and centrifuged for 2 minutes at 2,000 x g.
  7. 26 μL of neutralization buffer were added to each collection tube. A second elution was collected after repeating the same process one more time.

Method for His-tagged C-RP Purification using Ultrafree® devices

Solutions
  • Lysis buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM immidazole, pH 7
  • Binding buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM immidazole, pH 7
  • Wash buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 20 mM immidazole, pH 7
  • Elution buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 250 mM immidazole, pH 7
  • 1 mg/mL Lysozyme stock
  • Benzonase
Procedure
  1. Express recombinant proteins in Escherichia coli.
  2. Prepare at a 10X concentration using lysis buffer. (Pellet 100 mL of culture, resuspend in 10 mL of lysis buffer.)
  3. Add lysozyme to a concentration of 0.1 mg/mL and Benzonase® nuclease to reduce the viscosity of the lysate.
  4. Clarify the lysate by centrifugation (10 min at 10,000 x g).
  5. Add 200 μL of 50% resin slurry to the Ultrafree®-MC device. Spin the device for 1 min at 500 x g to remove any residual fluid of the resin.
  6. Equilibrate the resin with 500 μL of binding buffer and centrifuge for 2 minutes at 500 x g.
  7. Add 500 μL of the clarified lysate to the resin and incubate for 30 min with light agitation.
  8. Spin the device at 500 x g for 10 min to remove the unbound lysate proteins.
  9. Wash the resin with 500 μL of wash buffer and incubate for 5 minutes with agitation. Spin the device for 5 minutes at 500 x g.
  10. Repeat the washing step two more times.
  11. Add 250 μL of elution buffer and mix for 5 minutes.
  12. Spin the device for 1 min at 500 x g to recover the purified protein

Results

Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/ultrafiltration-jan2017-figure1-500px.jpg
Figure 1 shows the SDS-PAGE analysis for rabbit IgG purified using an Ultrafree®-MC centrifugal device. Starting material contained approximately 14 mg of total protein, with an estimated 1.5–2 mg IgG content. The total amount of purified IgG was 1.2 mg and 1.1 mg (estimated by OD280) on two devices processed in parallel. This method can be useful for monitoring the titer of antigen-specific antibodies after immune activation, or whenever small amount of IgG is needed.


Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/ultrafiltration-jan2017-figure2-500px.jpg
Figure 2 shows the SDS-PAGE analysis of His-tagged protein purified using an Ultrafree®-MC devices, with Ni-NTA agarose and BD Talon™ resin. From 500 μL of lysate, two recombinant proteins were purified, 32-35µg of CRP (a 26 kDa, high-expressing protein) and 8 μg of RT66 (a 66 kDa, low-expressing protein). This method is applicable to recombinant protein purification, antibody purification, and immunoprecipitation.



Affinity Purification with Buffer Exchange and Concentration: Amicon® Pro System

Traditional centrifugation-based affinity purification presents certain challenges. Batch processing of resins directly in microcentrifuge tubes is subject to sample loss during aspiration. Spin columns alleviate this concern; however, due to their limited volume capacity (typically 0.5 mL), the protocol is tedious, requiring multiple spin cycles during the wash and elution steps. Moreover, while most purification workflows require additional sample neutralization, buffer exchange, and/or concentration prior to downstream analysis, no single traditional centrifugation device is capable of performing all steps in the purification process.

The Amicon® Pro purification system is an adaptable centrifugal device that couples affinity-based spin column purification with downstream sample concentration and buffer exchange. By condensing the protein preparation workflow, the Amicon® Pro system eliminates the need for multiple sample transfers, thereby minimizing protein loss.

Amicon® Pro Protocol for Affinity Purification

This protocol was derived from the study described in the application note, The Amicon® Pro purification system: condensing the protein purification workflow into one centrifugal device. The method was designed for the affinity purification of GST- or His-tagged recombinant protein (from 0.5 mL of lysate).

NOTE: For all protocols using the Amicon® Pro device, all steps, with the exception of binding reactions, are spin-based. A swinging bucket rotor is required for all steps with the exception of the reverse spin.

Add 100 µL Resin (50% Ni-NTA agarose or glutathione agarose slurry) and 1.5 mL Wash buffer to exchange reservoir. Spin 1000 x g for 1 min.

  1. Add 0.5 mL sample and mix with resin by pipetting. Incubate with upright agitation in an orbital shaker for 1 h. [Note: end-over-end mixing may be less effective, because residual resin on the wall and cap may not be collected efficiently.]
  2. Spin 1000 x g for 1 min to clear unbound species.
  3. Add 1.5 mL Wash buffer. Spin 1000 x g for 1 min.
  4. Attach the Amicon® Ultra 0.5 mL filter (10k MWCO).
  5. Add 1 mL Elution Buffer and mix with resin. Incubate for 5 min.
  6. Spin at 4000 x g for 15 min.
  7. Add 1.5 mL desired buffer. Spin 4000 x g for 15 min to exchange buffer and concentrate.
  8. Recover purified protein from the Amicon® Ultra 0.5 filter by reverse spin.

Results

33% faster, less hands-on time. The Amicon® Pro device offered a significant reduction in overall processing time (33%, a savings of 63 min) while also minimizing hands-on time by decreasing the spin steps (3 to 1, in each case) required during the wash, elution, concentration, and buffer exchange phases. SDS-PAGE showed that, despite fewer washes in the Amicon® Pro protocol, high yield and purity were maintained (See figure below).

Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/high-yield-amicon.jpg

High yield and protein purity from Amicon® Pro purification compared to two traditional affinity purification schemes. In each case, 100 µL settled resin was used to purify His-CRP from 0.5 mL E coli lysate. A representative gel shows the various fractions derived during purification using the three bind-wash-elute methods: Amicon® Pro device, 0.5 mL spin column, and slurry batch purification method using Ni-NTA resin. Detailed methods can be found in the application note


Elution without dilution. For the elution phase, a single one-minute spin was sufficient to release >90% of bound protein (See figure below). Moreover, when the included Amicon® Ultra 0.5 mL filter was attached, the sample underwent simultaneous concentration during the elution step. The fraction’s final concentrated volume was, on average, 45 µL following a 15 minute spin. By combining these two steps, the Amicon® Pro device further eliminated the tedious requirement of determining protein content across the various eluted fractions before concentrating.

Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/protein-elution.jpg

Protein elution without sample dilution. 100 µL Ni-NTA agarose resin and 0.5 mL His-CRP lysate were added to Amicon® Pro or traditional devices (n = 3) and incubated for 1 hour. After clearance and washing, proteins were eluted from spin devices, either once in 1.0 mL Elution Buffer (Amicon® Pro) or 3 times with 500 µL Elution Buffer (Competitor spin column). While three elution steps were required for the smaller spin columns, a single spin in the Amicon® Pro device was sufficient to recover > 90% of affinity-captured protein in an average of 45 µL final volume*.

*For these samples, the included Amicon® Ultra 0.5 mL filters were attached, so that the samples underwent simultaneous concentration during the elution step. The fraction’s final concentrated volume was, on average, 45 µL following a 15 minute spin.

Protocols for Antibody Labeling, Purification and Concentration:

Click on links to view protocols.

Protein Labeling and Purification with Amicon® Pro Centrifugal Filters

Amicon® Pro Purification System for Antibody LabelingCommercially available protein labeling kits provide researchers with the flexibility to customize their own immunoassay detection panels. However, because many protocols require buffer exchange prior to and following antibody labeling, dialysis-based workflows are time-consuming and subject to significant protein loss at multiple points of sample transfer.

For removing unincorporated label in protein labeling applications, centrifugal devices are a fast, convenient, high-recovery alternative to gel filtration.

Small-Scale (25 μg – 200 μg) Antibody Labeling Using the Amicon® Pro Centrifugal Filter

Significantly higher yields can be achieved from antibody labeling by combining the labeling step with the clean-up step in the all-in-one Amicon® Pro system. The Amicon® Pro purification system is an adaptable centrifugal device coupling affinity purification with downstream sample concentration and buffer exchange.

Two attributes of the Amicon® Pro device make it a convenient device for preparing pure, labeled antibody:
  1. First, the device enables highly efficient buffer exchange via diafiltration with simultaneous sample concentration in a single 15 minute spin.
  2. Second, the entire workflow can be performed within a single device, reducing the potential for sample loss.
For details on the Amicon® Pro Antibody Labeling application, please visit our dedicated Antibody Labeling page, as well as our publication (Read our 2015 paper in Journal of Immunological Methods)

Read below for a brief summary of the protocol for using the Amicon® Pro device for small–scale antibody labeling.

NOTE: All steps, with the exception of binding reactions, are spin-based. A swinging bucket rotor is required for all steps with the exception of the reverse spin.

Optional: Pre-wet device using 0.5 mL TBS-T. Spin at 1000 x g for 1 min.
  1. Load antibody solution into the Amicon® Pro device as follows:
    For dilute antibodies (< 1 mg/mL), attach an Amicon® Ultra 0.5 mL filter (10k MWCO) to the base of the Amicon® Pro device. Load antibody (up to 1 mL) into the exchange device. Spin 4000 x g for 15 min.
  2. For concentrated antibodies (≥ 1 mg/mL), load sample (up to 100 µL) into an Amicon® Ultra 0.5 mL filter (10k MWCO). Attach filter to the base of the Amicon® Pro device.
  3. Prepare a 1.5 mL reaction cocktail containing dye in appropriate reaction buffer. Add cocktail to the exchange reservoir.
  4. Spin 4000 x g for 15 min.
  5. Optional: Incubate sample for an additional 30 minutes at room temperature, if necessary for optimal labeling efficiency. Read our publication for more details.
  6. Add 1.5 mL phosphate-buffered saline (PBS) ± Na Azide to the exchange reservoir.
  7. Spin 4000 x g for 15 min to exchange buffer and concentrate.
  8. Recover labeled antibody from the Amicon® Ultra 0.5 filter by reverse spin.

Sample Results of Antibody Labeling Protocol

Traditional 0.5 mL centrifugal  diafiltration deviceAmicon® Pro Purification System
Sample clean-up 45 min 15 min
Biotinylation reaction 120 min 30 min
Remove free biotin, buffer exchange, concentrate 45 min 15 min
Total protocol time 3.5 hours 1 hour
Antibody recovery 40% 72%

Table: Comparison of biotinylation workflows with respect to time and antibody recovery.

To compare the functional performance of antibodies labeled using traditional centrifugal diafiltration and using the Amicon® Pro method, their detection ability was compared with that of a species/anti-species GAPDH detection pair. Using similar amounts of primary antibody for each blot, both biotin/SA pairs demonstrated the same, if not slightly better sensitivity, than the species/anti-species detection pair (See figure below).

Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/anti-gapdh.jpg
Anti-GAPDH, biotinylated using the Amicon® Pro system, was used to successfully detect GAPDH in Western blots. Aliquots of EGF-stimulated A431 cell lysate (0.25-4 µg) were resolved by SDS-PAGE, transferred onto Immobilon®-P membrane, and probed for β tubulin in the SNAP i.d.® 2.0 system using the various indirect detection pairs. In each case, the same quantity of primary (GAPDH-specific) antibody was used.



Clean-up of Antibody Labeling Reactions Using Amicon® Ultra Filters

Using a standard Amicon® Ultra centrifugal filter, a few rounds of diafiltration can efficiently remove unincorporated label from an antibody labeling reaction without diluting the sample. Use the protocol below to remove unreacted fluorescent labels using centrifugal ultrafiltration devices.

Removal of unreacted fluorescent label from antibody conjugation reaction
  1. Load 1 mg of fluorescent-labeled antibody solution in 2 mL into two Amicon® Ultra-4 mL 10 kDa MWCO devices and centrifuge at 4,000 x g for 10 minutes.
  2. Redilute retentates (about 50 μL each) to 2 mL with water and centrifuge again. Repeat this step twice.
  3. After each centrifugation, save samples of the retentate and filtrate for SDS-PAGE analysis and quantitation using UV-Vis spectroscopy.

Results

Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/clean-up-amicon-ultra-filters-fig1-fig2.jpg

Figure 1 shows the SDS-PAGE gel of FITC-labeled BSA before and after each of four diafiltration cycles. Unincorporated FITC is visible in the starting material and in the first filtrate. The majority of the free label is removed following one ultrafiltration cycle; free FITC is virtually undetectable after subsequent filtration cycles. Fluorescence measurement data in Figure 2 confirm this result. The free FITC signal is reduced by ~ 80% after the first ultrafiltration, and further reduced to 98% removal after subsequent filtration cycles. This demonstrates the viability of ultrafiltration as an alternative to gel filtration for clean-up of protein labeling reactions.


Rapid, Ultrafiltration-based Method for Purification of Monoclonal Antibodies Using the Centricon® Plus-70 Device

Introduction

Monoclonal antibodies (MAb) continue to gain importance as therapeutic and diagnostic agents for many cancers. The process of screening hybridoma libraries for candidate MAbs is both time consuming and labor intensive. Once a hybridoma cell line expressing the desired MAb is established, a bench-scale purification methodology (e.g., 50 to 500 mL) must be developed to produce sufficient MAb for further characterization. A traditional method for purifying MAbs involves these steps:
  1. Clarifying the hybridoma supernatant by centrifugation
  2. Concentrating the Mab by ammonium sulfate precipitation
  3. Resolubilizing the MAb and desalting using dialysis
  4. Additional purification using protein A/G affinity chromatography
  5. Desalting and buffer exchange using dialysis
This process typically requires several days to complete and can be labor-intensive when evaluating multiple MAbs in parallel. Ultrafiltration-based MAb purification, on the other hand, is easier, faster (2–3 hours versus 2 days), and yields higher recoveries.

Method

The method involves clarification of the hybridoma supernatant by microfiltration using a Stericup vacuum filter cup, followed by concentration using ultrafiltrationwith a Centricon® Plus-70 device:
  1. Add 200 mL supernatant to Centricon® Plus-70 Device (100K MWCO) as follows:
    1a. Load 65 mL of supernatant to the device
    1b. Centrifuge (20–30 min)
    1c. Discard filtrate
    1d. Repeat steps 1a-1c two more times until all the supernatant has been loaded
  2. Invert device and spin to collect sample (2 min)
  3. Load on protein-G column
  4. Further purify on protein A/G beads
  5. Desalt and buffer-exchange using ultrafiltration

Results

Ultrafiltration was successfully used to purify an anti c-myc antibody secreted by the hybridoma clone 9E102 (See figure 1 below). The purified MAb performed comparably to traditionally/commercially prepared MAbs in Western blotting and ELISA assays (See figure 2 below). Our data show that the combination of microfiltration and ultrafiltration is a rapid method for Mab purification. Additionally, MAb purified on Montage® centrifugal columns with Protein G media is comparable to the commercially available MAb in purity, activity and cost.


Figure 1

Figure 2

Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/ultrafiltration-centricon-figure1-v2.jpg Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/ultrafiltration-centricon-figure2.jpg

Figure 1. Purification of anti c-myc antibody using ultrafiltration.
C: clarified supernatant; Tr: Supernatant precipitated with ammonium sulfate and dialyzed using 10K MWCO membrane (Spectrapor, Rancho Dominguez, CA, USA); UF: Supernatant concentrated by ultrafiltration on Centricon®-Plus 70 device.

Figure 2. Upper panel shows activity comparison measured in a Western blot between Mab (derived from HEK 293 cells) purified using ultrafiltration versus traditional methods. Three-fold serial dilutions of HEK 293 cell nuclear extracts were run on 4-12% SDS-PAGE gels and transferred to Immobilon®-P membrane. Blots were probed with the indicated primary antibodies and secondary anti-mouse alkaline phosphatase conjugate (MilliporeSigma). The antibodies were detected with alkaline phosphatase substrate and imaged on a Kodak imager. Bottom panel shows activity comparison measured in an ELISA. HEK 293 cells were grown on 96-well plates. After fixation and epitope retrieval by heating for 10 minutes in a microwave oven, cells were permeabilized with 1% saponin (MilliporeSigma) in PBS and 2% normal donkey serum (Jackson Immunoresearch, West Grove, PA) and treated with serial dilutions of the indicated purified MAbs. The cells were then washed and treated with goat anti-mouse HRP conjugate antibody (MilliporeSigma). The reactions were developed using a SureBlue™ TMB HRP substrate (KPL, Gaithersburg, MD, USA). The readings were measured on a SpectraMax® plate reader (Molecular Devices, Sunnyvale, CA, USA).


References
  1. Saha K, Case R, Wong PK. J Immunol Methods 1992; 151(1-2):307-308.
  2. Evan GI, Lewis GK, Ramsay G, Bishop JM. Mol Cell Biol 1985; 5(12):3610-3616.



Small-scale Antibody Purification With Buffer Exchange and Concentration: Amicon® Pro System

If an antibody must be purified from a small sample volume (0.5-10 mL), then the Amicon® Pro Purification System is recommended. Because this centrifugal device enables purification, buffer exchange, and concentration to be performed without transferring the sample or diluting it, the risk of antibody loss is minimized. The following method was developed for affinity purification of antibodies (0.5 mL sample) using protein A/G agarose resins in the Amicon® Pro System.

Method

  1. Add 200 µL Protein A/G Resin (50% slurry) and 1.5 mL Wash buffer to exchange reservoir. Spin 1000 x g for 1 min.
  2. Add 0.5 mL sample and mix with resin by pipetting. Incubate with upright agitation in an orbital shaker for 1 h. [Note: end-over-end mixing may be less effective, because residual resin on the wall and cap may not be collected efficiently.]
  3. Spin 1000 x g for 1 min to clear unbound species.
  4. Add 1.5 mL Wash buffer. Spin 1000 x g for 1 min.
  5. Attach the Amicon® Ultra 0.5 mL filter (10k MWCO).
  6. Add 1 mL Elution Buffer and mix with resin. Spin 4000 x g for 10 min.
  7. Add 1.5 mL final buffer of your choice. Spin 4000 x g for 15 min to exchange buffer and concentrate.
  8. Remove Amicon® Ultra filter and place into filtrate collection tube.
  9. Recover purified antibody from the Amicon® Ultra 0.5 mL filter by reverse spin.

Results

IgGs from rabbit serum (Cat. No. S20-100ML were purified on Protein A or Protein G agarose, using either the Amicon® Pro System (10kDa MWCO, Cat Nos. ACK5010PA and ACK5010PG) or traditional spin columns. Purified IgGs were then buffer-exchanged and concentrated, either in the same Amicon® Pro devices or transferred from spin columns to separate Amicon® Ultra devices.

When the Amicon® Pro System was used, the process required 6 pipetting steps/spins, as compared to 11 pipetting steps/spins required for traditional spin columns and concentrators. As shown in the figure below, antibody recovery remained unchanged. 

Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/rabbit-serum-chart.jpg
Antibody purification from rabbit serum using Amicon® Pro System and traditional spin columns (n=3)


Rapid Antibody Concentration with Amicon® Ultra Centrifugal Filters

Introduction

Purification protocols for immunoglobulins generally include affinity binding to Protein A or G chromatography media, washing unbound proteins, and eluting with a low pH buffer. The resulting purified antibodies are often too dilute for downstream studies or long-term storage. Dialysis is often necessary to concentrate the antibody and/or exchange the buffer into one that preserves protein activity. Ultrafiltration achieves the same result faster, with up to 99% immunoglobulin recovery and one-step salt removal (see Concentration, Desalting, and Buffer Exchange protocol).

Method and Results

Purification and concentration of Rabbit IgG with Prosep® A and Prosep® G Montage® Antibody Purification Kits and Amicon® Ultra-15 mL centrifugal filters.

A. Purification of IgG using Montage® Kits
  1. Equilibrate the Prosep® A or Prosep® G resins with 10 mL of binding buffer and spin for 5 min at 500 xg
  2. Pre-clarify the serum sample by passing through a 0.22 µm Steriflip® GP filter.
  3. Dilute the sample 1:1 with binding buffer A and centrifuge the device at 100-150 x g for 20 min.
  4. Wash the spin column with 20 mL of binding buffer A to remove unbound contaminants by centrifuging the column for 5 min at 500 x g.
  5. Elute the samples by adding 10 mL of elution buffer B2 into a fresh centrifuge tube containing 1.3 mL of neutralization buffer.
  6. Measure the total protein concentration of the sample using the Direct Detect® Spectrometer or other appropriate concentration method, such as UV absorbance at 280 nm.
B. Concentration of IgG using Amicon® Ultra-15 30kDa MWCO devices.
  1. Add the neutralized eluted fraction into Amicon® Ultra 15 mL 30 kDa MWCO device.
  2. Centrifuge for 10 to 20 min at 4000 x g in a swinging bucket rotor or 5000 x g in a fixed angle rotor until the sample has reached the desired concentration.
Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/rapid-antibody-concentration-fig1-fig2.jpg

Figure 1 shows a decrease in the retentate volume proportional to the increase in antibody concentration. A 20-minute centrifugation step resulted in >95% recovery of immunoglobulins. Figure 2 shows an SDS-PAGE gel of purified rabbit IgGs before and after ultrafiltration. The data demonstrate the suitability of Amicon® Ultra devices for concentrating purified IgG.

Protocols for Specialized Applications:

Click on links to view protocols. 

A Simple Strategy for Protein Enrichment Using Ultrafiltration

Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/serial-fractionation.jpg
Click image to enlarge.
The human plasma proteome is a vast resource for biomarker discovery and investigation. Traditionally, gel filtration chromatography has been used to fractionate protein solutions based on size1,2. However, gel filtration is laborious, limited by sample size, and time-consuming. It also dilutes the sample significantly. These limitations can be overcome by ultrafiltration, which has been reported3–5 as a sample preparation method for preparing low molecular weight (<10 kDa) fractions for biomarker analysis. Here, Amicon® Ultra devices were used to purify cytochrome c from a mixture containing 10% fetal bovine serum.

Method

In this experiment, ultrafiltration devices were used to enrich both low molecular weight (LMW) and high molecular weight (HMW) fractions from serum. Using a serial filtration approach (Figure 1), proteins were fractionated through decreasing molecular weight cutoff (MWCO) in Amicon® Ultra 4 mL devices ranging from 100 kDA to 10 kDa MWCO.

Results

Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/serial-enrichment-fig2-fig3.jpg 

This serial enrichment strategy enabled protein compartmentalization and improved throughput as compared to direct filtration using a lower MWCO device (Figure 2, above). Samples prepared using the enrichment stragegy also showed improved purity compared to traditional direct filtration (Figure 3, above). The data show that serial ultrafiltration is a viable approach for size-based protein enrichment.

References

  1. Werner MJ. Chromatogr 1966; 25(1):63-70.
  2. Kent UM. Methods Mol Bio 1999; 115:11-18.
  3. Forssmann WG, et al. J Chromatogr A 1997; 776:125-132.
  4. Plebani M, et al. Pancreas 2002; 24:8-14.
  5. Sobrinho LG, et al. Clin Endocrinol (Oxf) 2003; 58:686-690.



Urine Concentration with Amicon® Ultra Centrifugal Filters

Accurate measurement of specific proteins in urine is important for diagnosing and managing diseases. However, the concentration of urine proteins is often below the detection limit of clinical tests. Amicon® Ultra-4 mL 10 kDa MWCO devices can be used to concentrate urine samples prior to clinical laboratory analyses.

Method

Enrichment of free immunoglobulin light chains (Bence-Jones proteins) in urine samples using Amicon® Ultra-4 mL 10 kDa MWCO filters.
  1. Determine the total protein in a 24-hour urine specimen.
  2. Fill Amicon® Ultra-4mL device with 4 mL of urine.
  3. Centrifuge at 3400 x g for 30–45 minutes (until approximately 25–50 μL of concentrated sample is obtained). This represents a 160-fold increase in concentration of the original sample.
  4. Insert a pipette tip into the bottom of the filter unit and withdraw the concentrated sample.
  5. Perform agarose electrophoresis on the concentrate to quantitate light chains and identify other proteins. Determine the percentage of light chains with respect to the total number of components in the urine. Then multiply the percentage of light chains by the total 24-hour protein concentration (grams per 24-hour volume).
  6. Perform immunofixation electrophoresis on the concentrate to identify light chains.
Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/urine-method.jpg
Results of electrophoretic resolution of free immunoglobulin light chains (Bence-Jones proteins) in urine samples concentrated using Centricon® (C) and Amicon® Ultra (A) centrifugal concentration devices. The dark bands at ~25 kDa in each lane represent these proteins.

Additional Notes:

  1. Normal heterogeneous immunoglobulins may also be seen in urine concentrate with immunofixation electrophoresis. This “ladder effect” is comprised of microheterogeneous light chains. Bence-Jones proteins may be within this ladder. To verify the presence of Bence-Jones proteins requires additional analysis by two-dimensional electrophoresis.
  2. If there is excess antigen, dilution of the concentrate will be required until equilibrium is achieved between the antigen (Bence-Jones protein) and the antibody.

Related Products:

  1. Amicon® Ultra devices can also be used to concentrate serum, plasma and cerebrospinal fluid for similar analyses. A concentration of approximately 20 mg/mL is required in order to detect free light chains from diseased patients by agarose electrophoresis. Detection by immunofixation electrophoresis is 10 times more sensitive than by agarose electrophoresis.
  2. Static concentrators (Minicon® devices) are also available for concentration of Bence-Jones protein in urine.

Acknowledgements

Research using Amicon® Ultra devices for urine concentration in this protocol was conducted by Mark Merchant, Ph.D. at Helena Laboratories, Beaumont, TX.

References

  1. Tietz, N. Clinical Guide to Laboratory Tests, 2nd ed. Philadelphia: Saunders, WB; 1990;362–363.
  2. Kahns L. Clinical Chemistry 1991;37: 1557–1558.
  3. Cleveland Clinic homepage. Accessed July 2002. www.clevelandclinic.org/myeloma/ DiagnosisAndTreatmentOf MultipleMyeloma.html
  4. Christenson RH, et al. Clinical Chemistry 1983;29(6):1028–1030.
  5. Christenson RH, and Russell ME. Clinical Chemistry 1985;31(6):973.


Two-Dimensional Electrophoresis Sample Preparation with Amicon® Ultra Centrifugal Filter

Two-dimensional electrophoresis (2DE) is one of the most commonly used methods in proteome analysis. Briefly, proteins are separated by their isoelectric point (first dimension separation) followed by SDS-PAGE separation by molecular weight (second dimension separation). The salts and ionic detergents routinely used in sample preparation are not compatible with 2DE; the combination of high salt concentration and low protein content interferes with isoelectric focusing. One way to concentrate proteins is by acetone or TCA precipitation. However, this method has two key disadvantages:
  • Some proteins become insoluble and cannot be resolubilized in IPG buffer
  • Many salts become insoluble in acetone and precipitate along with the proteins

Method

Forty grams of bovine liver were blended in an industrial blender in 50 mL 50 mM Tris-HCl, 150 mM NaCl, containing protease inhibitor cocktail. The lysate was cleared by centrifugation, aliquoted, and stored at -20 °C. Protein concentration of 5 mg/μL was determined by BCA protein assay using bovine serum albumin standard.

For 2-DE analysis of the neat lysate, 150 μL of the extract was mixed with 50 μL IPG rehydration buffer and used to rehydrate an IPG strip. For 2-DE analysis of lyophilized lysate, the dried sample was resolubilized in 200 μL of IPG rehydration buffer, cleared by centrifugation, and used to rehydrate an IPG strip.

For 2-DE analysis of acetone-precipitated proteins, 150 μL liver lysate was mixed with 4 volumes of cold acetone and incubated for 1 h at -20 °C, followed by centrifugation at 12,000 rpm for 10 min. The supernatant fluid was decanted and the pellet was resuspended in 200 μL of rehydration buffer.

For 2-DE analysis of liver lysate prepared by ultrafiltration, 1 mL lysate was dispensed into an Amicon® Ultra-4 10,000 MWCO centrifugal device. The device was spun at 3000 x g for 40 min until approximately 100 μL of concentrated volume was remaining in the device. Nine-hundred microliters of 8 M urea, 2% CHAPS solution was added to the device, and centrifugation was repeated for 40 min. The concentrated proteins were immediately transferred to a microcentrifuge tube and the volume was adjusted to 1 mL with IPG rehydration buffer (8 M urea, 2% CHAPS, 0.002% bromophenol blue, 40 mM DTT, and 0.5% Pharmalyte). One-hundred fifty microliters of reconstituted ultrafiltrate were used to rehydrate an IPG strip in 200 μL total volume.

Results

Bovine liver lysate was prepared by homogenization in isotonic buffer with protease inhibitors. The protein concentration in the extract was determined to be 5 mg/mL. In order to successfully analyze the sample by 2-DE, it was estimated that approximately 0.75 mg of total protein was required, and thus 150 mL of the lysate was prepared in 200 mL volume by addition of 50 mL IPG rehydration buffer (20% of total volume).

2-DE of neat lysate resulted in poor separation and resolution, opresumably because of the high salt concentration and low concentration of chaotropic agents (Figure 1A). To achieve better separation, various methods of sample concentration were attempted. First, the sample was lyophilized and resuspended in IPG rehydration buffer. The resulting 2-D gel was streaky with no well-defined spots and with protein retained at both ends of the IPG strip (Figure 1B).

Acetone precipitation is another method for protein concentration. Figure 1C shows a 2-D gel of acetone-precipitated liver lysate. Because the salt content was greatly reduced compared to Figures 1A and 1B, a satisfactory separation was achieved. While protein recovery appeared to be complete, evaluating this assumption required an alternative method for protein concentration and desalting/ Ultrafiltration (UF) is a well-known method for protein concentration and desalting. Bovine liver proteins were concentrated by centrifugal UF. In this process, proteins are retained and thereby concentrated, while the salt concentration does not change and is the same in the retentate and the ultrafiltrate. However, because of the reduced volume of the retentate, the salt-to-protein ratio in the retentate is lower, and the sample is easily rendered suitable for 2-DE after dilution with IPG buffer.

Figure 1D shows a 2-D gel of liver proteins prepared by UF. As expected, the separation was successful, resulting in multiple well-defined spots on the 2-D gel.
Figure 1A
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Figure 1B
Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/electrophoresis-Fig1b.jpg

Figure 1C
Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/electrophoresis-Fig1c.jpg

Figure 1D
Merck:/Freestyle/BI-Bioscience/PSP/PSP-images/electrophoresis-Fig1d.jpg

Figure 1. 2-DE gel of 0.75 mg (in 200 μL total volume) bovine liver lysate prepared (A) by mixing proteins with IPG rehydration buffer (B); by lyophilization and resuspension in IPG rehydration buffer (C); by acetone precipitation (C), and (D) by UF. Results show that using ultrafiltration to concentrate a 2-DE lysate sample yields better resolution and higher protein recovery without detectable loss of low-molecular-weight species.

Reference:

  1. Chernokalskaya et al. Ultrafiltration for proteomic sample preparation. Electrophoresis 2004, 25, 2461–2468.


Purification of Serum Peptides for MALDI Mass Spectrometry with Amicon® Ultra Centrifugal Filters

Biomarkers play an essential role in the drug discovery and development process. They provide powerful clues to genetic susceptibility, disease progression and drug response. Serum is a key source of putative protein biomarkers. One major impediment to discovering new biomarkers is the presence of salts, proteins and lipids in plasma or serum, which interferes with peptide analysis by mass spectrometry. Multiple protocols can be used to extract and enrich peptides from tissues and body fluids:
  • After protein precipitation, fractionate by reversed-phase chromatography over C18 resin
  • Acetonitrile precipitation of large proteins, while enhancing the solubility of smaller proteins and peptides
  • Ultrafiltration for preparing low molecular weight fractions for biomarker analysis1–4
Ultrafiltration, in combination with solid phase extraction (SPE) on C18 resin is a convenient and efficient method for serum peptide purification. This approach provides more peptides for mass spectrometry analysis when compared to acetonitrile precipitation method. In addition, filter-aided sample preparation (FASP), which was originally developed using our Microcon® centrifugal filters5, has recently been optimized for use on Amicon® Ultra centrifugal filters6.

One advantage of FASP is that samples can be buffer-exchanged and concentrated through multiple spins without drying/precipitation concerns. Furthermore, FASP was adapted to 96-well plate format using our MultiScreen® Filter Plates7. Adaptation to the 96-well format permitted fast processing of biological material and opened possibilities of automating the FASP process in the future.

Methods

I. Preparation of serum peptides
  1. Dilute 1 to 4 mL of serum, plasma or cell lysate with 10% acetonitrile or with 10-20 mM tris HCL buffer pH 7.5 in a 1:1 ratio.
  2. Load the sample in Amicon® Ultra-4 10K NMWL centrifugal devices, and centrifuge in a swinging bucket rotor for 15-30 minutes at 3000 x g.
  3. Collect the filtrate.
  4. If sample contains acetonitrile, place it in a speed vacuum to evaporate the reagent; if not, continue directly with step 5.
  5. Acidify 10 μL of the filtrate sample using 5 μL of 1% TFA.
  6. Following the ZipTip® μC18 pipette tips protocol, desalt concentrate and clean the sample.
  7. Elute the sample directly onto a MALDI target with 2 μL of ∂-cyano-4-hydroxycinnamic acid matrix (5 mg/mL in 50% acetonitrile, 0.1% TFA). Note: If acetonitrile was added to the serum prior to the filtration, briefly centrifuge the samples in a Speed Vac® centrifuge to remove solvent before ZipTip® purification.
II. Peptide analysis by mass spectrometry using the filtrate of Amicon® Ultra 4 mL 10kDa centrifugal ultrafilters
  1. Acidify peptide-containing ultrafiltrates from cell lysates or human serum with 1% TFA and concentrate on ZipTip® μC18 or ZipTip® strong cation exchange pipette tips following the procedure outlined in the user guide.
  2. Overlay the sample with 1 μL of ∂-cyano-4-hydroxycinnamic acid matrix (5 mg/mL in 50% acetonitrile, 0.1% TFA) and analyze on MALDI mass spectrometer.
III. Preparation of Serum Peptides by Acetonitrile Precipitation (control samples)
  1. Dilute the serum sample as indicated in the previous protocol I.1) by adding 10% acetonitrile in a 1:1 ratio (v:v),
  2. Load the sample in a 15 mL centrifuge tube and centrifuge the samples to precipitate the proteins.
  3. Collect the supernatant and place it in a speed-vac centrifuge to evaporate the acetonitrile.
  4. Resuspend the sample in 0.1% TFA, desalt and concentrate with ZipTip® μC18 pipette tips.
  5. Perform co-elution directly onto a MALDI target with 2 μL of ∂-cyano-4-hydroxycinnamic acid matrix (5 mg/mL in 50% acetonitrile, 0.1% TFA).

Results

Amicon® Ultra-4 30K NMWL centrifugal devices can be used for preparing serum peptides for analysis by high resolution mass spectrometry. When compared to unprocessed rat serum, data quality is improved after removing large proteins by acetonitrile precipitation (Figure 1A, B, below). The number and quality of MALDI-TOF detected peptide peaks was significantly improved using the ultrafiltered serum (Figure 1B, below).

Data quality was further improved by incorporating reverse phase chromatography in the sample preparation protocol, either alone, or in combination with acetonitrile precipitation and ultracentrifugation (Figure 2, below). Here, the ZipTip® μC18 pipette tip served as a convenient and efficient tool for micro-scale sample preparation prior to mass spectrometry. The highest signal, signal-to-noise ratio and number of detected peptides was achieved with samples prepared using all three methods (Figure 2D, below).

This approach can be used directly in combination with ZipTip® μC18 pipette tips for peptide identification by MS/MS, or as a first step prior to further surface-mediated enrichment using SELDI-TOF methods. These protocols may be applicable to other low molecular weight markers, such as drugs and metabolites.

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