Western Blotting Protocols

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Standard vs. Rapid Immunodetection Procedures

There are two types of protocols for immunodetection: Standard and rapid.

StepStandard ImmunodetectionRapid Immunodetection
Block the membrane 1 hr None
Incubate with primary antibody 1 hr 1 hr
Wash the membrane 3 x 10 min 3 x 5 min
Incubate with second antibody 1 hr 30 min
Wash the membrane 3 x 10 min 3 x 5 min
Add substrate 5 min 5 min
Total time 4 hr 5 min 2 hr 5 min

Standard Immunodetection Methods Include the Following Steps:

  1. Blocking unoccupied membrane sites to prevent nonspecific binding of antibodies
  2. Incubating the membrane with primary antibody, which binds to the protein of interest
  3. Washing to remove any unbound primary antibody
  4. Incubating the membrane with a conjugated secondary antibody, which binds the first antibody
  5. Washing to remove any unbound secondary antibody
  6. Incubating the membrane with a substrate that reacts with the conjugated secondary antibody to reveal the location of the protein
Rapid immunodetection eliminates the blocking step and reduces the time necessary for the washing and incubation steps. The rapid immunodetection method works well to quickly visualize higher abundance proteins. Standard immunodetection, however, offers higher sensitivity and requires less optimization for new sample types.


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Standard Immunodetection Method

Application
Standard immunodetection is performed on blotted proteins directly after electrotransfer. (If the membrane was dried after transfer, thoroughly wet the blot for 1 minute in methanol if using PVDF or Milli-Q water if using nitrocellulose before proceeding to immunodetection.)

The unoccupied membrane binding sites on the wet blot are blocked with optimized reagents. The drawbacks of this method are the need for blocking and the total time requirement of over 4 hours. The advantage is that standard immunodetection may require less optimization for new sample types.

Required Solutions
  • Primary antibody (specific for protein of interest)
  • Secondary antibody (specific for primary antibody), labeled with alkaline phosphatase or horseradish peroxidase.
  • Substrate appropriate to the enzyme conjugate (HRP or AP).
  • Appropriate wash buffer: Phosphate-buffered saline (PBST): 10 mM sodium phosphate, pH 7.2, 0.9% (w/v) NaCl, up to 0.1% Tween-20 detergent, TBST: 10 mM Tris, pH 7.4, 0.9% (w/v) NaCl, up to 0.1% Tween-20.
  • Blocking solutions: bovine serum albumin (BSA), 0.2%–5% (w/v), Tween-20,(0.05–0.1%), non fat dry milk (0.5–5%), Casein(1%).
  • Milli-Q water
Required Equipment
  • Shallow trays, large enough to hold blot
  • Glass plates
  • Plastic wrap (e.g., Saran™ film), freezer bag, or sheet protector
  • Autoradiography film and cassette
  • Dark room
  • Autoradiography film processing equipment
Set Up
  1. Dilute the primary antibody in the blocking solution to the desired working concentration.
  2. Dilute the secondary antibody in the blocking solution to the desired working concentration.
Note: Enough solution should be prepared to allow for 0.1 mL of antibody solution (primary and secondary) per cm2 of membrane.


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Western Blotting Protocol

  1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer the protein to the membrane (electroblotting)

    Products to prepare the gel:
    • OmniPur SDS (Cat No. 7910)
    • OmniPur Acrylamide (Cat No. 1150)
    • Tris (Cat No. 648313-250GM)
    • OmniPur Amonium Persulfate (Cat No. 2300)
    • OmniPur TEMED (Cat No. 8920)
    • OmniPur Water (Cat No. 9820)
    • Tween 20 (Cat No. 655204-100ML)
  2. Wash the membrane twice with distilled water (Cat No. 4.86505.0500). If desired, stain the membrane with Ponceau Red solution for 5 minutes to visualize protein bands. (Stock solution: 2% Ponceau S in 30% trichloroacetic acid and 30% sulfosalicylic acid; dilute 1:10 for use.) Rinse the membrane in water until protein bands are distinct and mark the position of the molecular weight markers with a ballpoint pen or pencil. The Ponceau Red stain will be washed off the membrane during the blocking step. Note: Do not let the blot dry out at any step through development, as this will cause an increase in background staining.

    Block the blotted membrane in freshly prepared TBS (Cat No. 524750-1EA) and/or PBS (PBS Tablets 524650-1EA) containing nonfat dry milk (3–5% ) (see note on blocking) for 30–60 minutes at room temperature with constant agitation. A maximum blocking time of 2 hours at room temperature should not be exceeded since staining artifacts will appear. If longer blocking times are required, the membrane should be kept at 4°C.

    Note on blocking: Soak the blotted membrane in freshly prepared blocking reagent, PBS/3% nonfat dry milk (15gms nonfat milk in 500mLs PBS (PBS Tablets 524650-1EA) for 30 minutes to 2 hours at room temperature with constant agitation. Membranes may also be blocked with PBS/3% nonfat dry milk overnight at 4°C. Generally, phospho-antibodies require blocking reagents diluted in TBS (Cat No. 524750-1EA) rather than PBS. If previous Western blots had high backgrounds, try a different blocking buffer. Oter blocking reagents which can be used include a) 3–5% Nonfat dry milk/0.05–0.1%Tween, b) Tween-20 (0.05–0.2%) (Cat No. 655204-100ML) in PBS or TBS and c) 0.2–5% BSA fraction V (Cat No. 2910) in PBS or TBS. Generally, maximum blocking time should not exceed 2 hours at room temperature or proteins can be exchanged from the membrane.

  3. Dilute the primary antibody to the recommended concentration/dilution in fresh blocking solution (TBS and/or PBS (PBS Tablets 524650-1EA) /3% nonfat dry milk). Incubate the membrane in the primary antibody solution for 1 to 2 hours at room temperature or overnight at 4°C with agitation.

  4. Wash the membrane three times for 3 to 5 minutes each with either water or TBS and/or PBS containing 0.05% Tween-20 (Cat No. 655204-100ML).
  5. Incubate the membrane in the secondary antibody reagent of choice for 30 minutes to 1 hour at room temperature or overnight at 4°C with agitation. For a mouse monoclonal antibody, a goat anti-mouse HRP-conjugated antibody is recommended, for a rabbit polyclonal antibody, a goat anti-rabbit HRP-conjugated antibody is advisable.

  6. Wash the membrane five times for 3 to 5 minutes each time with either water or TBS and/or PBS (PBS Tablets 524650-1EA) containing 0.05% Tween-20 (Cat No. 655204-100ML).

    Note: Tween-20 detergent has the potential to strip low affinity primary antibodies, and therefore the membrane is briefly washed to improve the background. If the membrane has been washed with water, a final wash step for 3 to 5 minutes in TBS and/or PBS (PBS Tablets 524650-1EA)containing 0.05% Tween-20 (Cat No. 655204-100M) should follow.

  7. Perform the detection of proteins using detection system of choice, (e.g., enhanced chemiluminescence (ECL))

    Note: If Tween-20 is not exhaustively removed by washing the membrane with either water, TBS and/or PBS, the Tween-20 could react with ECL reagent, and there may be an increase in overall membrane staining resulting in a black film.

    Western Blotting Protocol


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Detection Substrates

Chromogenic Detection
  1. Prepare the substrate according to manufacturer’s instructions.
  2. Place the blot in a clean container and add substrate to completely cover the surface of the membrane. Incubate for 10 minutes with mild agitation or until signal reaches desired contrast.
  3. Rinse the blot with Milli-Q water to stop the reaction.
  4. Store the blot out of direct light to minimize fading. Blot may be stored dry.
Chemiluminescent Detection
  1. Follow manufacturer’s instructions.
  2. Prepare the substrate according to manufacturer’s instructions.
  3. Place the blot in a container and add substrate to completely cover the membrane. Incubate for 1 minute.
  4. Drain excess substrate.

    Note: A cut-to-size sheet protector or a freezer bag can also be used.

  5. Gently smooth out any air bubbles. In a dark room, place the wrapped membrane in a film cassette.
  6. Place a sheet of autoradiography film on top and close the cassette.
  7. Expose film. Multiple exposures of 15 seconds to 30 minutes should be done to determine the optimum exposure time; 1 to 5 minutes is common.
Fluorescent Detection
Required Equipment:
  • Proteins blotted onto Immobilon-FL transfer membrane and probed with antibodies
  • Mylar® wrap
  • Fluorescent imaging equipment
The following is a general protocol for fluorescent immunodetection. For optimal results, refer to manufacturer’s protocol provided with the reagents.

Note: If using chemifluorescent reagents, follow reagent manufacturer’s directions.

  1. Prepare the substrate according to manufacturer’s instructions.
  2. Place the blot in diluted fluorescent dye-labeled secondary antibody solution and incubate for 1 hour with gentle agitation.
  3. Wash the blot with wash buffer 3–5 times for 5 minutes each.
  4. Place the blot onto a piece of clean filter paper to dry.
  5. If using a wrap, use Mylar. Do not use Saran® wrap because it quenches the fluorescence.
  6. Image the blot using an appropriate fluorescence scanner.


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Rapid Immunodetection Method

Application
Rapid immunodetection takes advantage of the fact that antibodies cannot bind to the hydrophobic (non-wetted) surface of the Immobilon-P transfer membrane, but will bind to a protein immobilized on the membrane. Rapid immunodetection is compatible with both chromogenic and chemiluminescent substrates. The major advantage of rapid immunodetection is that blocking is not required, saving time and eliminating the risks involved (Mansfield, 1994). The method can be used on both dry and wet Immobilon PVDF membranes, enabling the analysis in about 2 hours, as opposed to over 4 hours for the standard method.

Important: When using a dry membrane, the blot must be thoroughly dry before beginning rapid immunodetection (refer to Membrane Drying Methods).

Required Solutions
  • Primary antibody (specific for protein of interest)
  • Secondary antibody (specific for primary antibody), labeled with alkaline phosphatase or horseradish peroxidase
  • Substrate appropriate to the enzyme conjugate
  • Phosphate buffered saline (PBS) (PBS Tablets 524650-1EA): 10 mM sodium phosphate, pH 7.2, 0.9% (w/v) NaCl
  • Antibody dilution buffer: 1% (w/v) BSA (bovine serum albumin), 0.05% Tween-20 detergent (Cat No. 655204-100ML)
  • Methanol, 100%
  • Milli-Q water
Required Equipment
  • Shallow trays, large enough to hold blot
  • Plastic wrap (e.g., Saran), freezer bag, or sheet protector
  • Autoradiography film and cassette
  • Dark room and autoradiography film processing equipment
Set Up
  1. When using a dry membrane, dry the blot completely (refer to ‘Membrane Drying Methods’ protocol). Do not re-wet the blot in methanol.
  2. Dilute the primary antibody in dilution buffer to the desired working concentration.
  3. Dilute the secondary antibody in dilution buffer to the desired working concentration. Note: Enough solution should be prepared to allow for 0.1 mL of antibody solution (primary and secondary) per cm2 of membrane.
Antibody Incubations
  1. Place the blot in the primary antibody solution and incubate with agitation for 1 hour. The solution should move freely across the surface of the membrane.
  2. Place the blot in PBS and wash for 5 minutes.
  3. Place the blot in the secondary antibody solution and incubate with agitation for 30 minutes.
  4. Place the blot in PBS (PBS Tablets 524650-1EA) and wash for 5 minutes. Repeat twice with fresh buffer.
  5. Proceed with chromogenic, chemiluminescent or fluorescent protein detection as described under the ‘Standard Immunodetection Method’ protocol.


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High Salt Wash to Remove Persistent Background

Required Solutions:
High salt buffer: PBS (PBS Tablets 524650-1EA) or TBS buffer supplemented with 0.5 M NaCl and 0.2% SDS

Procedure:
  1. Follow the Standard Immunodetection Method through primary and secondary antibody incubations.
  2. After the secondary antibody incubation, place the blot in the high salt buffer and incubate for 30 minutes with gentle shaking.
  3. Rinse blot with Milli-Q water and proceed with chromogenic, chemiluminescent or fluorescent protein detection as described in the Standard Immunodetection Method.
High Salt Wash to Remove Persistent Background
Adding 0.5M NaCl and 0.2% SDS to the wash buffer reduced the background considerably on the Immobilon-PSQ membrane, which as a high protein binding capacity.


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Membrane Stripping Protocols

Two protocols for removing antibodies from blots are presented below. The first is applicable to any chemiluminescent substrate system and uses a combination of detergent and heat to release the antibodies. The second is commonly used for applications where antibodies have to be separated from an antigen and employs low pH to alter the structure of the antibody in such a way that the binding site is no longer active. Neither method will remove the colored precipitates generated from chromogenic detection systems (e.g., BCIP, 4CN, DAB and TMB). However, it is still possible to analyze the blot with another antibody specific to a different target protein.

Important: The blot should not be allowed to dry between rounds of immunodetection. Any residual antibody molecules will bind permanently to the membrane if it is allowed to dry.


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Re-Blot™ Plus

Western Blot Recycling Kit
The Re-Blot™ Plus Western Blot Recycling Kit contains specially formulated solutions that quickly and effectively remove antibodies from Western blots without significantly affecting the immobilized proteins.

Advantages of Re-Blot™ Plus Western Blot Recycling Kit
  • No pungent-smelling ß-mercaptoethanol is contained in the Antibody Stripping Solution.
  • Antibody stripping is done at room temperature. No heating of blots is required.
  • Blots can be stripped of antibodies in approximately 15 minutes at room temperature.
  • Blots may be reused in 25 minutes.
Application
The Re-Blot™ Plus Western Blot Recycling Kit (Cat. No. 2500) is effective for removal of antibodies from Western blots that have been developed with chemiluminescence or radioactive iodine or other isotopes.

It is not recommended for stripping colorimetric substrates (TMB, DAB, 4-chloronapthol, etc.), as it is not possible to effectively remove substrates that precipitate at the reaction site. The Re-Blot™ Plus Western Blot Recycling Kit should be used only for qualitative purposes until it has been established by comparative blot analysis that stripping does not quantitatively affect a given antigen.

Kit Components
  • Mild Antibody Stripping Solution (10x) – (1 container, 50 mL). (May be purchased separately as Cat. No. 2502.)
  • Strong Antibody Stripping Solution (10x) – (1 container, 50 mL). (May be purchased separately as Cat. No. 2504.)
Storage
Kit components should be stored at 4°C upon arrival. Product is stable for 3 to 6 months after receipt. If Antibody Stripping Solution crystallizes upon storage, it may be re-dissolved with gentle warming at 37°C before use.

Note: To prevent reagent degradation secure the cap tightly upon storage. Avoid extended exposure to air.

Selection of Reagent
Re-Blot™ Plus Mild Stripping Solution gives good results on both nitrocellulose and PVDF membranes. However, Re-Blot™ Plus Strong Stripping Solution will perform better when membranes with high signal are to be stripped or when Re-Blot™ Plus Mild treatment is not sufficient.

# of Strips
or Blots
Amount of 10X Antibody
Stripping Solution
Amount of
Distilled Water
(Cat No. 4.86505.0500)
Resulting Amount of 1x
Working Stripping Solution
1 Strip 400 µL 3.6 mL 4 mL
5 Strips 1 mL 9.0 mL 10 mL
1 Blot (7 x 10 cm) 2 mL 18.0 mL 20 mL

Preparation of Reagents
  • Dilution of Antibody Stripping Solution
  • Immediately before use: Dilute Antibody Stripping Solution (Mild or Strong) 10x with distilled water (Water Cat No. 4.86505.0500) to obtain a 1x solution. If Antibody Stripping Solution contains crystals, warm gently at 37°C until crystals have dissolved completely. Prepare enough solution to allow free movement of strips or blots during incubation, typically 4 mL per strip or 20 mL per standard blot.
Use the following chart for suggested volumes of stripping or blocking solution.

Assay Protocol
Note: The blots or individual strips that are to be re-used should be prepared for stripping immediately after their first usage. If stripping cannot be performed right away, membranes can be wrapped in plastic wrap and stored moist in PBS at 4°C. Do not store blots in dry form.

  1. Blot Stripping. Fill plastic tray with appropriate amount of 1x Antibody Stripping Solution (see Preparation of Reagents for suggested quantities).
  2. Using tweezers or forceps, submerge blot or blot strips in stripping solution. Incubate with gentle mixing for 15 minutes at room temperature.

    Note: It may be necessary to increase the stripping incubation time when using blots that have been stripped previously. Simply increase the stripping time by 5 to 10 minutes, if needed.

  3. Blocking. Fill a clean plastic tray with an equal amount of blocking buffer. Conventional blocking buffers such as 20 mM Tris Hcl, pH 8.0; 150 mM NaCl; 0.1% Tween 20 (Cat No. 655204-100ML); 5% dry milk or similar are appropriate.
  4. Wash blots two times 5 minutes each with blocking buffer.
  5. The blot is now ready for reprobing with antibodies. It is suggested that users employ their own proven protocol and chemiluminescent detection method.


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Stripping by Heat and Detergent

Required Equipment and Solutions
  • Stripping solution: 100 mM 2-mercaptoethanol, 2% (w/v) SDS, 62.5 mM Tris-HCl, pH 6.7
  • Phosphate buffered saline (PBS PBS Tablets 524650-1EA): 10 mM sodium phosphate, pH 7.2, 0.9% (w/v) NaCl
  • Shallow tray, large enough to hold the membrane
Procedure
  1. In a fume hood, place the blot in stripping solution and incubate with agitation for 30 minutes at 50°C
  2. Place the blot in buffer and agitate for 10 minutes. Repeat with fresh buffer.
  3. (Optional) Repeat the initial detection protocol (omitting the primary antibody step) to make sure that the antibodies have been inactivated or stripped from the membrane.
  4. Place the blot in buffer and agitate for 10 minutes.
  5. Proceed to the blocking step for the next round of immunodetection.


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Stripping by Low pH

Required Equipment and Solutions
  • Stripping solution: 25 mM glycine-HCl, pH 2, 1% (w/v) SDS
  • Phosphate buffered saline (PBS PBS Tablets 524650-1EA): 10 mM sodium phosphate, pH 7.2, 0.9% (w/v) NaCl
  • Shallow tray, large enough to hold the membrane
Procedure
  1. Place the blot in stripping solution and agitate for 30 minutes.
  2. Place the blot in buffer and agitate for 10 minutes. Repeat with fresh buffer.
  3. Proceed to the blocking step for the next round of immunodetection.
Stripping by Low pH
Stripping and reprobing on Immobilon-P membrane using low pH and heat/detergent methods. (A) Membrane blots of rat liver lysate were probed with rabbit anti-ERK-1 antibody (1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (1:20,000) prior to visualization with Immobilon Western HRP Chemiluminescent Substrate. The blots were then stripped by (B) low pH or (C) heat/detergent and re-exposed to the secondary antibody and the Immobilon Western substrate to demonsrate complete removal of all antibodies. Blots stripped of anti-ERK-1 were then probed with a mouse anti-HSP70 antibody 1:10,000) and HRP- conjugated goat anti-mouse secondary antibody (1:5,000) and visualized as above. Blots stripped by (E) low pH and(F) heat/detergent were successfully re-probed and yielded results comparable to (D) membrane probed only with antibodies to HSP70.


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Peptide Inhibition/Competition

Use this protocol when using antibodies used with competing immunogens.

Method
  1. Determine the highest antibody dilution at which the optimal working dilution is used with the antibody in Western blotting.
  2. For neutralization, combine antibody with a fivefold (by weight) excess of blocking peptide in a small volume of TBS and/or PBS-milk (1 mL), or combine antibody with 10µM–25µM* concentration of peptide in final volume of incubation buffer (3% non-fat dry milk in TBS and/or PBS(PBS Tablets 524650-1EA). See the example peptide calculation below.
  3. Incubate the peptide and antibody together for 2 hours at room temperature or overnight at 4°C with agitation.
  4. Following the neutralization, dilute the antibody/ peptide mixture into the appropriate final working volume and proceed with the protocol for the desired test, and proceed with the Western blot procedure as usual.
Example
Antibody gives good positive results at 2 µg/mL. Normally nitrocellulose is incubated in a final volume of 10 mLs. Combine 20 µg of the antibody with 100 µg of the peptide in 1mL of P(T)BS-milk. Incubate. Add the 1mL of peptide/antibody to 9mL of P(T)BS-milk.

* Preparing a 1mM stock of peptide to be further diluted:

MW = Da – [(x–1)(18)]
Da = Daltons
X = number of amino acids in peptide sequence
18 = MW of H20

Example for Diluting a Peptide with a molecular weight of:

2136 Da = 2136 g/L/M
2136 g/L x 0.001 M
2.136 g/L/1 mM
2136 mg/1000 mL/1 mM

If peptide is packaged at 50 µg:

Peptide Inhibition/Competition

Dilute 50µg of peptide in 23 µl sterile water (Cat No. 4.86505.0500) (or other appropriate buffer) to make the peptide at a final concentration of 1 mM.


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Blot Storage

Preparation of Protein Blots for Long-term Storage
PVDF is a chemically resistant polymer with excellent long term stability. For blots that need to be stored for use at a later date, storage conditions are determined by the stability of the proteins bound to the membrane. While EMD Millipore recommends storage at low temperature, room temperature may be adequate for some proteins.

Required Materials
  • Dry blotted Immobilon PVDF transfer membrane
  • Two sheets of Whatman 3 MM paper
  • Two sheets of card stock or thin cardboard
  • Paper clips
  • Plastic bag
Procedure
  1. Place the dry blot between two clean sheets of Whatman 3 MM paper.
  2. Place the blot-filter paper sandwich between two sheets of card stock.
  3. Clip the stack together along the edges. The clips should not overlap the blot.
  4. Place the stack into a sealable plastic bag.
  5. Close or seal the bag.
  6. Store the blot at the desired temperature: 4°C For up to 2 weeks – 20°C For up to 2 months – 70°C For longer term storage.
Note: Blots stored in a freezer should not be subjected to mechanical shock, which can cause breakage of the membrane. The blot should be allowed to come to room temperature before removal from the plastic bag. Blots may also be stored wet at 4°C in a plastic bag, but a bacteriocide such as sodium azide should be added to prevent bacterial growth. The azide must be thoroughly washed out of the blot prior to use as it inhibits HRP activity.

Links for OmniPur Products: