Immobilon® Membranes, Sandwiches and Blotting Filter Paper

PVDF transfer membranes for Western blotting

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Immobilon® Membranes, Sandwiches and Blotting Filter Paper Clear Sorting & Filtering
Catalogue Numbericon Descriptionicon Filter Dimensions (cm x cm)icon Pack Sizeicon
IBFP0785CImmobilon Blotting Filter Paper, 7 x 8.4 cm sheet7 x 8.4100 Show Pricing & Availability
IBFP0813CImmobilon Blotting Filter Paper, 8.5 x 13.5 cm sheet8.5 x 13.5100 Show Pricing & Availability
IPFL00010Immobilon-FL PVDF, 0.45 µm, 26.5 cm x 3.75 m roll26.5 x 3751 Show Pricing & Availability
IPFL10100Immobilon-FL PVDF, 0.45 µm, 10 x 10 cm sheet10 x 1010 Show Pricing & Availability
IPFL20200Immobilon-FL PVDF, 0.45 µm, 20 x 20 cm sheet20 x 2010 Show Pricing & Availability
IPSN07852Blotting Sandwich Immobilon-P membrane interleaved with blotting paper 7 x 8.4 cm7 x 8.420 Show Pricing & Availability
IPSN08132Blotting Sandwich Immobilon-P membrane interleaved with blotting paper 8.5 x 13.5 cm8.5 x 13.520 Show Pricing & Availability
IPVH00010Immobilon-P Membrane, PVDF, 0.45 µm, 26.5 cm x 3.75 m roll26.5 x 3751 Show Pricing & Availability
IPVH07850Immobilon-P Membrane, PVDF, 0.45 µm, 7 x 8.4 cm sheet7 x 8.450 Show Pricing & Availability
IPVH08100Immobilon-P PVDF 0.45 µm 8 x 10 cm sheet8 x 1010 Show Pricing & Availability
IPVH08130Immobilon-P PVDF 0.45 µm 8.5 x 13.5 cm sheet8.5 x 13.510 Show Pricing & Availability
IPVH09120Immobilon-P Membrane, PVDF, 0.45 µm, 9 x 12 cm sheet9 x 1210 Show Pricing & Availability
IPVH10100Immobilon-P Membrane, PVDF, 0.45 µm, 10 x 10 cm sheet10 x 1010 Show Pricing & Availability
IPVH15150Immobilon-P Membrane, PVDF, 0.45 µm, 15 x 15 cm sheet15 x 1510 Show Pricing & Availability
IPVH20200Immobilon-P Membrane, PVDF, 0.45 µm, 20 x 20 cm sheet20 x 2010 Show Pricing & Availability
IPVH304F0Immobilon-P Membrane, PVDF, 0.45 µm, 26 x 26 cm sheet26 x 2610 Show Pricing & Availability
ISEQ00010Immobilon-PSQ Membrane, PVDF, 0.2 µm, 26.5 cm x 3.75 m roll26.5 x 3751 Show Pricing & Availability
ISEQ07850Immobilon-PSQ Membrane, PVDF, 0.2 µm, 7 x 8.4 cm sheet7 x 8.450 Show Pricing & Availability
ISEQ08100Immobilon-PSQ PVDF 0.2 µm 8 x 10 cm sheet8 x 1010 Show Pricing & Availability
ISEQ08130Immobilon-PSQ PVDF 0.2 µm 8.5 x 13.5 cm sheet8.5 x 13.510 Show Pricing & Availability
ISEQ09120Immobilon-PSQAU10435 PVDF 0.2 µm 9 x 12 cm sheet9 x 1210 Show Pricing & Availability
ISEQ10100Immobilon-PSQ Membrane, PVDF, 0.2 µm, 10 x 10 cm sheet10 x 1010 Show Pricing & Availability
ISEQ15150Immobilon-PSQ Membrane, PVDF, 0.2 µm, 15 x 15 cm sheet15 x 1510 Show Pricing & Availability
ISEQ20200Immobilon-PSQ Membrane, PVDF, 0.2 µm, 20 x 20 cm sheet20 x 2010 Show Pricing & Availability
ISEQ26260Immobilon-PSQ Membrane, PVDF, 0.2 µm, 26 x 26 cm sheet26 x 2610 Show Pricing & Availability

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    Documentation

    Brochure

    Title
    Western Blotting Tools

    Technical Info

    Title
    Protein Dot Blotting using Immobilon-P

    Data Sheet

    Title
    Immobilon Transfer Membranes: For superior protein and nucleic acid blots

    Posters

    Title
    Low Background Membrane for Fluorescent Protein Detection in Western Blotting

    References | 23 Available | See All References

    Reference overviewApplication
    Mobile Phase Preparation for UHPLC: Membrane Filtration Method Affects System Performance and Leaching of Extractable Impurities
    Subodh Kulkarn(1), Jesmi George(2) and Vivek Joshi(2) (1) Millipore India Pvt. Ltd., Bioscience Division, 50A, 2nd Phase, Ring Road, Peenya, Bangalore, India 560058 (2) Millipore Corp., Bioscience Division, 17 Cherry Hill Drive, Danvers, MA 01923
    LCGC 2010

    Show Abstract Full Text Article
    Quantitation of Protein on gels and blots by infrared fluorescence of Coomassie blue and fast green
    Luo S., Wehr N.B., Levine R.L.
    Analytical Biochemistry:350 (2006):233-238 2006

    Immunoblotting (Western)
    Pre-B-cell colony-enhancing factor is a secreted cytokine-like protein from the human amniotic epithelium.
    Ognjanovic S, Ku TL, Bryant-Greenwood GD.
    Am J Obstet Gynecol. 2005 Jul;193(1):273-82 2005

    Western Blotting
    Role of the Small Heat Shock Proteins in Regulating Vascular Smooth Muscle Tone
    McLemore E.C., Tessier D.J., Thresher J., Komalavilas P., Brophy C.M
    J. Am. Coll. Surg. 2005, Vol 201 (1):30-36 2005

    A high-affinity reversible protein stain for Western blots
    Antharavally B.S., Carter, B., Bell, P.A., Mallia K.
    Analytical Biochemistry 2004,Vol 329:276-280 2004

    Biochemical analysis of GABA receptor subunits alpha 1, alpha 5, beta1 beta2 in the hippocampus of patients with Alzheimer's disease neurophathology
    Rissman, R.A., Mishizen-Eberz A.J. N.,Wolfe, C.B.B., DeBlas A.L., Miralles C.P., Ikonomovic M.D., armstrong D.M.
    Neuroscience 120 (2003) 295-705 2003

    Western Blotting
    Proteomics reveals protein profile changes in doxorubicin treated MCF7 human breast cancer cells
    Cheng S.T., Pan T, L., Tsai Y.Ch, Huang C. M.
    Cancer letters 2002. vol 181:95-107 2002

    Towards proteome-wide production of monoclonal antibody by phage display.
    Bin Liu, Lan Huang, Carina Sihlbom, Al Burlingame and James D. Marks.
    J Mol Biol. 2002 Feb 1;315(5):1063-73 2002

    Mass Spectrometry Sample Prep
    Characterization of retinoic acid receptor-deficient keratinocytes.
    Goyette Philippe; Chen Chang Feng; Wang Wei; Seguin Francois; Lohnes David(a)
    Journal of Biological Chemistry v 275 pg 16497-16505 June 2, 2000 2000

    Protection of renal inner medullary epithelial cells from apoptosis by hypertonic stress-induced p53 activation
    Dmitrieva Natalia(a); Kultz Dietmar; Michea Luis; Ferraris Joan; Burg Maurice ; Journal of Biological Chemistry v 275, pg 18243-18247 June 16, 2000
    Journal of Biological Chemistry v 275, pg 18243-18247 June 16, 2000 2000

    FAQ

    QuestionAnswer
    Can I strip Immobilon-PSQ? No, because of the increased surface area and tighter pore size of this membrane it is very difficult to effectively strip this membrane.
    Should I prewet MultiScreen Immobilon-P plates before use? Yes. Use 15 ul of 70% ethanol or methanol to prewet the Immobilon P membrane. This membrane is hydrophobic and requires a prewet to allow liquid to pass through the membrane.
    What is the thickness of the Immobilon PSQ? Immobilon PSQ is 200 microns thick.
    What is the smallest size of a protein or peptide which binds to the Immobilon-PSQ? For the PSQ we do not suggest that they go lower than 2 kDa for the protein size. To help promote the transfer of the smaller proteins the methanol concentration of the transfer buffer can be increased to 20% (w/v) and the SDS lowered to 0.01%. The strength of the electric field can also be reduce by 50% to increase the proteins contact time with the membrane. Standard stains can be used such as Coomasie Blue and Ponceau. Keep in mind that some of the stain such as Coomasie Blue are not reversible.
    How many times can I strip and reprobe Immobilon-P? While 2-3 times is probably the limit, it is difficult to give an absolute number in regards to stripping and reprobing. This is because there are many factors to consider when it comes to protein binding to PVDF and primary antibody affinity to these proteins. Different proteins will have varying degrees of affinity to PVDF. This is based on factors discussed in our Protein Blotting Handbook (see TP001; Protein Binding section). One round of stripping may remove one protein and leave another intact. The same could occur when using different primary antibodies. Different antibodies will have varying affinities to different proteins. If carrying out several rounds of stripping and reprobing, one strategy might be to detect the least abundant proteins earlier leaving the higher abundant proteins later for detection.
    What is the difference between Immobilon–FL and Immobilon-P? Both membranes are made from PVDF. The difference in background fluorescence is due to proprietary modifications in the membrane manufacturing process.
    What fluorescence-based detection methods can be used with Immobilon-FL? Immobilon-FL can be used in Western blotting applications using either fluorescent dye-conjugated antibodies or chemi-fluorescence substrates. Western blots can be imaged in the visible or IR range. Single color detection or multiplexing for co-localization studies can be performed efficiently on Immobilon-FL Fluorescent proteins, e.g. green fluorescent protein, (GFP) or proteins tagged with GFP, blotted onto the membrane can be readily detected as well.
    What fluorescent dyes are recommended to use with Immobilon-FL? Immobilon-FL membrane exhibits very low background fluorescence over a broad range of wavelengths and can be used with all visible and infrared fluorescent dyes. Generally, dyes with larger Stokes shift (greater separation between absorption and emission wavelengths) are preferred in western blotting applications using fluorescence-based detection. NOTE: To prevent photo-bleaching, protect the membrane from light during secondary antibody incubations and washes until the membrane is ready to be scanned.
    How long does the fluorescent signal stay on the developed blot? The fluorescent signal from fluorescence-tagged antibodies will remain stable on the membrane for several months, or longer, when stored protected from light. The membranes may be stored dry or in PBS at 4ºC. The fluorescent signal from chemi-fluorescent substrates does not remain for long so blots using these substrates need to be scanned immediately.
    Do I need to dry Immobilon-FL blots before scanning? Drying the blot may enhance signal strength. NOTE: Do not wrap the blot in plastic/saran wrap while scanning. If you do need to wrap the membrane, Mylar should be used.
    Can Immobilon-FL be stripped and re-probed? Yes, Immobilon-FL can be stripped and re-probed like any other PVDF membrane, using standard detergent or acidic methods (refer to Millipore’s Protein Blotting Handbook). Keep the blot wet. If dried, rewet with methanol and then rehydrate before stripping the blot. Once the membrane has dried the stripping is ineffective. Alternatively, the blot can be photo-bleached to remove the fluorescence.
    If I need to write on an Immobilon-FL blot what marker should I use? A pencil should be used for writing/marking on the blot. Ink from most pens and markers may fluoresce or wash off and redeposit elsewhere on the blot. You can also use marker pens supplied by scanner manufacturers.
    What is the Rapid Immunodetection Method? Rapid Imunodetection is a method used with Immobilon-P that eliminates the blocking step by exploiting the inherently hydrophobic nature of the membrane. This hydrophobicity excludes the aqueous antibody solution from the internal pore structure of the membrane, thereby eliminating the need for a blocking step and greatly reducing the amount of washing required to remove excess immunoreagents.
    I want to strip my immunoblot and probe with another antibody. The rapid immunodetection protocol states that the membrane should be dry, but the stripping protocol states that the membrane should not be allowed to dry. What should I do? You dry the blot after transfer, and perform your rapid immunodetection protocol. Then, if you are planning to do rounds of immunodetection with other antibodies, you must keep the the blot wet after immunodetection is complete and continue with stripping off the first antibody. After that, you can proceed to the blocking step of the next round of immunodetection with the other antibody. Do not let the blot dry between rounds of immunodetection, otherwise, any residual antibodies from the first round will bind permanently to the membrane.
    Can the Rapid Immunodetection Method be used when probing for low abundant proteins? The Rapid Immunodetection Method is recommended for the quick detection of abundant proteins.
    Can the Rapid Immunodetection Method be used without prior drying of the membrane? The membrane has to be completely dry to assure low background with this method.
    Do any reagents used for immunodetection have to be treated prior to use? Depending on the product quality it may be necessary to filter solutions used for western blotting. It is advisable that solutions be prepared prior to use to avoid microbial contamination. Only high quality water such as Milli-Q water is recommended. Solutions can be filtered using Millex syringe filters, Steriflip filter units or Stericup units. Aggregates in the blocking and/or the secondary Antibody solution may result in a “speckled background.
    Does it matter whether PBS or TBS is used as the diluent with Immobilon Western Substrates? When using a detection system that utilizes alkaline phosphatase the use of TBS is recommended to reduce the phosphate concentration.
    What additional steps can be taken to reduce high background? One or more of the following steps may also be useful in reducing the background:
    • Assure that high quality water such as Milli-Q water is used for solution preparation.
    • Filter solutions used during Immunoblotting to remove aggregates.
    • Increase wash volumes and time as well as wash cycle repetitions.
    • Optimize blocking reagent.
    • Add 0.05% Tween 20 to all wash solutions to reduce protein-protein interactions. Avoid overexposure of the film by reducing the exposure time to one minute or less.
    What does negative staining (dark overall background with white bands on film) indicate? It may be the result of high secondary HRP conjugated antibody concentration. Loading less antigen on the SDS-PAGE gel and reducing the concentration of the secondary antibody by at least two fold should correct the problem.
    Which components can be optimized to reduce non-specific binding in a western blotting application using the Immobilon Western substrates? Non-specific binding may be optimized by any of the following:
    • Reducing the primary antibody concentration by at least two fold
    • Reducing the concentration of the secondary antibody and/or
    • Reducing the amount of protein loaded onto the SDS-PAGE gel
    I am seeing a weak signal in a western blot with the Immobilon Western Substrates. What can I do to increase signal intensity?
    • Ensure that a sufficient amount of protein was loaded onto the SDS-PAGE gel.
    • Check transfer efficiency by staining the membrane for total protein detection.
    • Select different antibodies with higher affinity. The concentration of the primary and secondary antibodies may be to low and requires further optimization.
    • Aliquot antibody to avoid repeat freeze and thaw cycles.
    • Check that all reagents are stored at the proper temperature to avoid degradation.
    • If using HRP Substrate, prepare fresh working reagent prior to substrate incubation.
    • The film exposure time can be increased.
    How can I increase antigen binding to the membrane? Tween-20 may wash the antigen of the membrane. Reduce or eliminate the use of Tween-20 except for the wash step following membrane blocking. Do not use SDS in the transfer buffer.
    Is re-blocking of the membrane required after complete stripping of a western blot? The membrane has to be blocked with the appropriate blocking agent prior to each immunodetection.
    Should any reagents be avoided with the use of Immobilon Western Substrates? When using the Immobilon Western HRP substrate, Azide should not be used in the buffers and reagents, as it inhibits HRP activity.
    I have been using Nitrocellulose for my Western Blots but am thinking of converting over to Immobilon-P PVDF membrane. Are there any differences in the protocol that I should know about? Protocols for Western Blotting with PVDF and Nitrocellulose are the same with a few exceptions. PVDF is hydrophobic and therefore should be prewet in methanol before it is used. Wet the membrane in methanol for 15 seconds. Membrane should uniformly change from opaque to semi-transparent. Carefully place the membrane in ultrapure water and soak for 2 minutes. Then carefully place the membrane in transfer buffer and let equilibrate for at least 5 minutes. Another change to note is that the SDS tolerances are not equivalent for the two membranes. The binding of protein to Immobilon P is much more sensitive to SDS levels. Too much SDS can inhibit the protein's ability to bind to the PVDF and can, in fact, help proteins already bound to the membrane to slip off. SDS levels should never exceed 0.05%.
    How should I store the Immobilon-P membrane? Prior to use the Immobilon-P membrane should be stored at room temperature. After use the membrane may be stored by allowing the membrane to dry, wrap in plastic wrap and store at 4º C. For short term (overnight) storage the membrane may be stored wet by wrapping in plastic and refrigerating.
    What's "blow-through", and how can I prevent it? Blow-through is a phenomenon seen in blotting applications where proteins move from the gel and through the membrane without binding to it. This is usually seen with low molecular weight proteins and can be minimized by gradually increasing the current or voltage during the transfer and optimizing the composition of the transfer buffer. Also, add a back-up sheet of membrane to check for any proteins that still may get through. It should be noted however, that for N-terminal sequencing applications, Immobilon-PSQ is the membrane of choice. The increased surface area and tighter pore size of this membrane result in better retention of small molecular proteins than Immobilon-P.
    Can I use Immobilon P for nucleic acids? Although, historically, there have been references that cited using Immobilon P with nucleic acids, Millipore strongly recommends that nucleic acids be transferred to either nitrocellulose or nylon membranes. Immobilon P is best suited for the transfer of proteins and there is an extensive body of literature within Millipore to support this application.
    Can I use Immobilon-P for dot or slot blotting of my proteins? Yes. If you are using a dot or slot blot manifold, first wet the Immobilon-P with 100% methanol, then rinse it with water, and then equilibrate in PBS or similar buffer. Place the Immobilon-P in the manifold, and add all samples before turning on the vacuum. If your manifold requires that one or more pieces of filter paper be placed under the transfer membrane, the filter paper that touches the membrane should also be wet with the buffer used to equilibrate the Immobilon-P.

    If you are not using a manifold, the Immobilon-P should be wet with methanol, rinsed with water, and equilibrated as above, and then placed on a piece of coarse filter paper (such as Whatman 3MM) that has been wet with the same buffer. It is helpful to place some dry paper, either Whatman 3MM or paper towels, beneath the wet Whatman in order to help reduce the tendency of the sample to spread laterally on the wet Immobilon-P. Following the spotting procedure (either with a manifold or without), for best results the membrane should be allowed to dry, then rewet with methanol and rinsed with water prior to blocking and immunodetection.
    What is CAPS buffer and when should I use it with Immobilon P? CAPS buffer, pH 11, is a buffer formulated without glycine that is commonly used in sequencing and amino acid analysis applications where residual glycine will interfere with interpretation of results. Because the pH is 11, this buffer can be used for the blotting of proteins with a pI >8.5. CAPS buffer is recommended for tank transfer systems. In semi-dry transfer, it may produce variable transfer efficiency.
    How can I elute protein off Immobilon-P? Several methods have been used depending on the nature of the study. Proteins can be eluted from the membrane by incubation at room temp. in 50 mMTris-HCL, pH 9, containing 2% SDS and 1% Triton X-100, followed by dialysis to remove most of the detergents. If the eluted protein is to be purified by HPLC, hydrogenated Triton-X-100 should be used as it will not contribute any UV absorbence to the chromatogram. If detergents in the protein solution are a problem, a 40% acetonitrile solution in 0.1 M ammonium acetate, pH 8.9 for 3 hours at 37oC can be used to elute the proteins. Lyophilization of the extract will remove the volatile solvents. If the eluted protein is to be purified by HPLC, hydrogenated Triton-X-100 should be used as it will not contribute any UV absorbence to the chromatogram.
    If I do a dot blot with Immobilon-P should I prewet the membrane? A dot blot can be done in two ways, via vacuum filtration with a dot blot apparatus or through intrusion with a tuberculin syringe. With the first method, the membrane must first be rendered hydrophilic using methanol (this allows the protein to adsorb onto the membrane) followed by a quick soak in water prior to assembly in the apparatus. When using the second method it isn't necessary to prewet with methanol. The user simply holds the syringe directly against the membrane and lets the pressure exerted from the syringe intrude the solution ( and protein) onto the membrane.
    How can I strip Immobilon-P? Processing a single blot with multiple antibodies requires the removal of the first antibody prior to the addition of a second antibody preparation. The stripping method should effectively disrupt the antigen-binding capacity of the antibody and solubilize it into the surrounding buffer. For most immunodetection procedures, it is also important to prevent spurious binding of the secondary antibody during stripping so that the enzyme conjugate will not contribute to background in subsequent rounds of detection. Finally, the stripping method should not cause a loss of antigens from the membrane. Two methods are provided for stripping an immunoblot. http://www.millipore.com/publications.nsf/docs/tp001en00protocols.
    My pre-stained protein markers disappear when I transfer my Western blot. What's happening? Some of the dyes used in pre-stained markers are very hydrophilic and the dyed protein may travel faster and penetrate more deeply into the membrane than the undyed protein. You can still check for the presence of your protein by transillumination of the membrane (after blot is dry, soak in 20% methanol for 5 minutes, and view translucent protein bands on a light box).
    Can I use the same stains with Immobilon-PSQ as I do with Immobilon-P? Yes, but the background will be higher than on Immobilon-P.
    Is there an effect of internal surface area on protein binding capacity? Yes, but the relationship between surface area and binding capacity is quite complex and is also influenced by the overall thickness of the membrane as well.
    My proteins bound just fine to Immobilon-P but not to Immobilon-PSQ under the same blotting conditions. Why? Blotting a large amount of protein from single bands or transferring at a high current (or voltage) setting may cause elution of enough protein to block the pores at the surface of the membrane. Additional protein that elutes from the gel will be unable to penetrate the membrane and will smear out across the surface of the blot, resulting in loss of the resolution achieved on the gel. To prevent this problem, reduce the current (or voltage) to 25% of the standard setting and then slowly ramp up to the maximum setting during the transfer period. This will allow eluting protein to effectively penetrate the membrane.
    Can I use Immobilon-PSQ for western applications involving small molecular weight proteins? Millipore recommends Immobilon-P for westerns because of higher backgrounds observed when detecting proteins on Immobilon-PSQ. The smaller pore structure of Immobilon-PSQ also tends to limit the accessibility of bound proteins to antibodies. If optimization of Immobilon-P for detection of your protein is unsuccessful, Immobilon-P can be substituted. To minimize the background, remember that Immobilon-PSQ binds about three times as much protein as Immobilon-P. The amount of blocking solution used should be adjusted accordingly. Also, the length and number of washes may need to be increased to effectively wash excess antibody reagents from the pores.
    What effect will the pore size and internal surface area for the Immobilon-PSQ membrane have on performance? We have studied the effect of the pore size and surface area of the Immobilon-PSQ membrane with respect to 3 different features: protein binding during blotting, protein elution and transillumination. The results are as follows.

    Protein binding during Blotting: Testing of Immobilon-PSQ using our standard blotting QC methods has shown that this membrane is effective in quantitatively binding proteins ranging from 14-120 KD. This is the key performance characteristic for which the membrane was originally developed.

    Protein Elution: the Immobilon-PSQ membrane has a smaller nominal pore size and a higher internal surface area than the Immobilon-P membrane. The membrane should, however, hold on to proteins and peptides better than the Immobilon P.

    Transillumination: The ability to visualize proteins by transillumination is a function of the thickness of the membrane and the distribution of the protein from the front to the back of the membrane. The Immobilon-PSQ has a smaller pore size than Immobilon-P but is thicker. Transillumination using Immobiolon-PSQ is not as sensitive as seen with Immobilon-P.
    I have just received the 0.2um Immobilon-PSQ membrane. Does the membrane require any special handling procedure? The Immobilon-PSQ can be handled in the same way as Immobilon-P. Prewetting in methanol and exchanging in MilliQ water are the only requirements prior to blotting.
    What effect will SDS have on the New Immobilon PSQ 0.2 um membrane? SDS interferes with the binding of protein to PVDF during transfer. Because this membrane is thicker than Immobilon P, there is a high probability that the protein will stick to the membrane. Because of the tighter pore size and increased thickness, the residence time of the protein within the membrane will favor its binding to the PVDF. If the protein is not retained well on the 0.2 um membrane, reducing the voltage or current may help as would lowering the SDS concentration ( only as a secondary step if reducing current/voltage does not work).
    What are Millipore's suggestions to reduce blow-through with the new 0.2um Immobilon PSQ? If blow-through is an issue, modify transfer conditions by reducing the voltage or current.
    How much protein do I need for sequencing? For most purposes, if the protein is visible by staining with Coomassie brilliant blue R, then there is enough protein present for sequence analysis. Check with your protein sequencing facility on the minimum requirements for their instrumentation.
    Can the 0.2 um Immobilon PSQ membrane be used for Immunodetection ? Immobilon- PSQ is recommended for transfering and detecting proteins of molecular weight less than 20 kD.
    Can I use ECL with Immobilon-PSQ ? Yes, standard chemilumniescent detection can be used with Immobilon-PSQ. Conditions used for the Immobilon-P will most likely require some optimization when switching to Immobilon-PSQ. Since the binding capacity of the Immobilon-PSQ is greater the mass of the blocking agent must be increased as well as the incubation time. You will also need to increase the wash time to ensure that the unbound anitbody is washed out of the tighter pores. Antibody concentrations may also need to be increased because the molecules are more likely to be distributed deeper into the pores. However modifying the blocking and wash steps as recommended may make adjusment of the antibody concentration unnecessary.
    Can I use the Rapid Immunodetection method with nitrocellulose? No, the surfactants used in the manufacture of nitrocellulose allow the membrane to wet out in all aqueous buffers. Immobilon-P is the membrane of choice for this method.
    What is the Rapid Immunodetection Method? Rapid Imunodetection is a method used with Immobilon-P that eliminates the blocking step by exploiting the inherently hydrophobic nature of the membrane. This hydrophobicity excludes the aqueous antibody solution from the internal pore structure of the membrane, thereby eliminating the need for a blocking step and greatly reducing the amount of washing required to remove excess immunoreagents.
    How can I transfer a range of high and low molecular proteins from my gel onto Immobilon-P without the small proteins being lost? Ramping of the current and optimization of the transfer buffer composition can be used together to give efficient blotting of proteins over a wide range of molecular weight. They may also be used separately to give the maximum transfer efficiency for either small or large proteins.

    1. Ramp - To help slow down small proteins, start the transfer at half the normal current (or voltage), and gradually increase the current (or voltage) during the transfer period to the maximum setting recommended. This measure allows the smaller proteins to travel more slowly through the electric field and have more residence time within the membrane. This step increases the chance that binding will occur. As the current is increased the larger proteins start to move out of the gel and onto the membrane as well.

    2. Optimize your transfer buffer - When optimizing your transfer buffer, it is important to realize that conditions favoring the transfer and binding of small proteins often have a negative effect on the transfer of large proteins. The converse is also true. The following rules are generally applicable. Most small proteins elute from a gel with little difficulty. To improve their binding to the membrane, SDS should be omitted from the transfer buffer and the methanol concentration should be increased to as high as 40%. For optimal blotting of large proteins, decreasing the methanol concentration to 10% or less and supplementing the transfer buffer with up to 0.05% SDS enhance their solubility and minimize their precipitation within the gel. Thus, they are better able to elute from the gel. Large proteins normally bind to the membrane very readily.
    I used the rapid immunodetection method with Immobilon-P and noticed higher backgrounds. What happened? The membrane wasn't completely dry. Try dipping the membrane in 100% Methanol after transfer and allow to dry in a hood for 15 minutes prior to exposure to the primary antibody.
    How can I make Immobilon-P transparent? Immobilon-P can be rendered transparent for use in densitometry studies, by immersion in a 3:2 solution of DMSO:ethanol. If the membrane has been allowed to dry it is necessary to prewet the membrane with methanol before starting this procedure.
    I transferred my protein to Immobilon-P and I can't find it. I've stained the gel and the membrane and nothing is there. What happened? If the pI of your proteins are greater than the pH of the transfer buffer, the proteins will travel in the opposite direction. The protein probably transferred into the running buffer. If you suspect that your protein has a high pI, try CAPS buffer and/or put membrane on both sides of the gel.
    What is the sensitivity of the transillumination method with Immobilon-P and how does it work? The sensitivity of transillumination is equivalent to Coomassie Blue staining. After drying the membrane, the PVDF is re-wet in 20% methanol and viewed in white light, or placed in a tray containing 20% methanol on a white light transillumination box.

    This method takes advantage of the change in refractive index of the rehydrated protein band to that of the water/methanol solution. The naturally hydrophobic Immobilon P membrane will not re-wet and, therefore can be visualized by the difference of refractive indices. The protein pattern appears translucent against the white membrane.
    Can Ponceau-S be used to stain proteins transfered to Immobilon-P? Ponceau-S can be used with Immobilon-P and is reversible. The stain produces pinkish bands on a light background. The stain can be removed by washing the blot with 0.1N NaOH.
    What are the important factors to consider when choosing a stain for use with Immobilon-P? The most important factor in choosing a stain is how the proteins on the blot are to be analyzed. For example, if immunodetection analysis will follow, non-reversible stains are not recommended. Non-reversible stains generally exhibit the best sensitivity but can interfere with or prevent further analysis of the proteins. Although less sensitive, reversible stains allow assessment fo the blot and then can be washed from the membrane. However, there is a risk that some proteins may undergo chemical modification during the process or be washed from the membrane.
    Is there is preferred side to use for Immobilon-P? No, Immobilon-P is manufactured to have no discernible difference in blotting performance on either side of the membrane.
    When using Immobilon-P in a slot blot unit, the membrane is sticking to the plastic part of the device under the slots. What can be done to keep the membrane from sticking and breaking? The sticking of the membrane is due to the type of plastic used in the blotting unit.

    One way to address this issue is to silicanize the surface of the plastic. This can be done using a product like Sigmacote (Sigma # SL-2). [The coating should be tested on a noncritical area of the unit, such as a small area on the exterior, to make sure that the solution is compatible with the type of plastic used in the device. The solvent for the silicone is heptane.] Pour a small amount of Sigmacote onto a kimwipe and then wipe across the surface that will be in contact with the membrane. Let the solvent dry off completely, and then wipe the surface with a clean kimwipe to remove any residue. Wash the unit thoroughly in Milli-Q water, and then proceed with the binding.

    Another potential problem is that the unit may be closed too tightly. To address this issue, close the unit with just enough pressure to obtain a seal; additional pressure is of no benefit.
    Can I use Immobilon-PSQ for all protein blotting applications? The Immobilon-PSQ membrane should not be used as a replacement for the Immobilon P unless Immobilon-P is not working in a particular protein transfer system. Immobilon-P has demonstrated superior capabilities in applications such as standard immunodetection, rapid immunodetection, standard ECL, rapid ECL and transillumination. The use Immobilon-PSQ membrane has been shown to be most applicable in the in immunoblotting of relatively small molecular weight proteins (less than 20,000 daltons).
    What is the protein binding capacity of Immobilon-P? The protein (BSA) binding capacity for the Immobilon P membrane is 131 micrograms per sq. cm. of membrane.
    Can ethanol be substituted for methanol in the Western Blot procedure? Yes, it is acceptable to replace the methanol with ethanol as long as you substitue it 1 for 1. So 20% methanol would be replaced with 20% ethanol.
    How does the protein binding capacity and protein retention of Immobilon-FL compare to Immobilon-P? Immobilon-FL’s protein binding capacity and protein retention are comparable to Immobilon-P.
    Is Immobilon-FL better than Nitrocellulose membrane for Fluorescence detection? Yes. Background fluorescence of Immobilon-FL is typically 2-5X lower than that of nitrocellulose, thus improving signal-to-noise ratio (sensitivity). Nitrocellulose membranes have other disadvantages. If allowed to dry out, they become brittle, tend to fracture and are difficult to handle. They are not recommended for stripping and re-probing. Nitrocellulose blots need to be scanned as soon as possible after detection, as diffusion of signal on a wet membrane may also occur.
    What is the level of detection sensitivity when using Immobilon-FL? Immobilon-FL exhibits the lowest background fluorescence of any blotting membrane thus improving the signal-to-noise ratio (sensitivity) of any fluorescent dye. The fluorescence signal is dependent on the fluorescent dye used, thus the ultimate sensitivity of detection is determined by the dye itself. With Immobilon-FL the sensitivity is not limited by the membrane, e.g., detection of proteins in the low picogram level has been observed with QDot® Nanocrystals fluorescent-tagged antibodies on Immobilon-FL.
    What are the recommendations for two-color western / multiplex detection? The two antibodies must be derived from different host species, so that they can be differentiated by secondary antibodies of different specificities. Before combining the two primary antibodies test their banding patterns on separate blots to know where to expect the bands. Use highly adsorbed cross-adsorbed secondary antibodies in two-color detection. Refer to the directions provided with the detection reagents for additional details.
    Are there specific recommendations on using 'low fluorescent’ reagents (buffers, blocking agents, etc.) with Immobilon-FL? Immobilon-FL has been tested with the most commonly-used blocking reagents and buffers such as TBST, PBST, dry milk, BSA, casein and buffers recommended by the fluorescence scanner manufacturers, e.g., Licor Odyssey buffers. All were found to be compatible with fluorescent detection on Immobilon-FL. However, as in any western blotting experiment, optimization of blocking reagents and buffers may be required, depending on the nature of the antigen, antibodies and fluorescent dyes used. The use of freshly prepared buffers is always recommended.
    Can I use Tween-20 in my buffers in western blotting with Immobilon-FL? Do not expose membrane to Tween-20 until after the blocking step is completed. Presence of Tween-20 during blocking may increase background fluorescence.
    Can Immobilon-FL be used with Millipore’s Rapid Immunodetection Method? Yes. Immobilon-FL can be used with the Rapid Immunodetection Method. However slightly higher background may be observed.
    Do I need to rinse the Immobilon-FL blotting membrane with water prior to blocking step, after wetting with methanol? It is not necessary to rinse the membrane with water. However, carry-over of methanol from the wetting step can reduce binding of blocking reagents to the membrane.
    Can Immobilon-FL be used with other detection chemistries (chromogenic, radioactive, and chemiluminescent)? Immobilon-FL is optimized for fluorescence-based detection applications. It can be used with other western blotting detection methods, e.g., chromogenic, radioactive and chemiluminescent, using standard protocols.
    How many times can I strip and reprobe Immobilon-P? While 2-3 times is probably the limit, it is difficult to give an absolute number in regards to stripping and reprobing. This is because there are many factors to consider when it comes to protein binding to PVDF and primary antibody affinity to these proteins. Different proteins will have varying degrees of affinity to PVDF. This is based on factors discussed in our Protein Blotting Handbook (see TP001; Protein Binding section). One round of stripping may remove one protein and leave another intact. The same could occur when using different primary antibodies. Different antibodies will have varying affinities to different proteins. If carrying out several rounds of stripping and reprobing, one strategy might be to detect the least abundant proteins earlier leaving the higher abundant proteins later for detection.
    I used the rapid immunodetection method with Immobilon-P and noticed higher backgrounds. What happened? The membrane wasn't completely dry. Try dipping the membrane in 100% Methanol after transfer and allow to dry in a hood for 15 minutes prior to exposure to the primary antibody.
    Can the 0.2 micron Immobilon PSQ membrane be used for Immunodetection in a western blot application? Yes. This membrane, because it is pure PVDF, will have the same compatibility with immunodetection systems as the 0.1 um system. As an advantage, the 0.2 um membrane is more open which will make it easier to block giving it lower non specific binding properties and make it more accessible to antibody probes. Proteins that were difficult to detect on the 0.1 um membrane may be more accessible on the 0.2 um Immobilon PSQ.
    I have been using Nitrocellulose for my Western Blots but am thinking of converting over to Immobilon-P PVDF membrane. Are there any differences in the protocol that I should know about? Protocols for Western Blotting with PVDF and Nitrocellulose are the same with a few exceptions. PVDF is hydrophobic and therefore should be prewet in methanol before it is used. Wet the membrane in methanol for 15 seconds. Membrane should uniformly change from opaque to semi-transparent. Carefully place the membrane in ultrapure water and soak for 2 minutes. Then carefully place the membrane in transfer buffer and let equilibrate for at least 5 minutes.
    Can I strip the antibodies from a western blot to probe for a different protein of interest? Yes. There are two commonly used methods:

    Method 1: Heat and detergent stripping method: Incubate the membrane for 30 minutes in 100mM 2-merceptoethanol/2% SDS/ 62.5mM Tris HCl pH 6.7 for 30 minutes (50oC) followed by two ten minute wash steps using PBST (PBS with 0.05% Tween-20) or TBST (TBS with 0.05% Tween-20).

    Method 2: Acidic stripping method: Incubate the membrane for 30 minutes at RT in 25mM Glycine (pH 2.0), 1% SDS for 30 minutes followed by two ten minute wash steps using PBST (PBS with 0.05% Tween-20) or TBST (TBS with 0.05% Tween-20).
    Can the colored precipitate formed after stripping the initial antibody in a western blot application be removed? The colored precipitate is permanent, but the blot can be analyzed with another antibody specific to a different protein.
    When using Immobilon Western reagents, my blots come out very dark with high background. What causes this and how can I eliminate it? High background and dark blots are often due to excess antibody. Due to the high sensitivity of Immobilon Western substrates, less primary and secondary antibody is needed for optimal detection. Typical antibody dilutions are 1:1,000 -1:20000 for primary and 1:20,000 – 1:200,000 for secondary antibodies. Reducing the amount of antibody will reduce and should eliminate the high background.
    When I convert to Immobilon Western substrates, should I first adjust the primary or secondary antibody dilution? The recommended approach is first to increase the dilution of the secondary antibody by 2 to 5 fold. Dilution of the Primary antibody can then be increased to further reduce the background and non-specific binding. For protocols where the optimal secondary antibody dilution has not been previously determined, an evaluation of performance is suggested with secondary antibody dilutions of 1:20,000, 1:50,000 and 1:100,000.
    I've completed my transfer to Immobilon-P, and now I see translucent areas on the membrane that correspond to the location of my wells or bands. Is there something wrong? No. When protein is transferred to Immobilon-P, the membrane becomes hydrophilic where the protein is bound. If the membrane is dried, or even partially dried, and then wetted with an aqueous buffer, the regions with bound protein will appear translucent.
    Can I use Coomasie Blue G-250 to stain my protein blots instead of Coomasie Blue R-250 (Brilliant Blue) which is recommended in your literature? Coomasie Blue G-250 will work, however, be aware that it will not have the same intensity and contrast as the R-250 will. The difference is caused by the way the stain interacts with the protein. G-250 doesn't give the same level of binding as the R-250. Bands may look light with the G-250, but keep in mind that the protein could still be there at a higher concentration than the stain would indicate.
    I want to strip my immunoblot and probe with another antibody. The rapid immunodetection protocol states that the membrane should be dry, but the stripping protocol states that the membrane should not be allowed to dry. What should I do? You dry the blot after transfer, and perform your rapid immunodetection protocol. Then, if you are planning to do rounds of immunodetection with other antibodies, you must keep the the blot wet after immunodetection is complete and continue with stripping off the first antibody. After that, you can proceed to the blocking step of the next round of immunodetection with the other antibody. Do not let the blot dry between rounds of immunodetection, otherwise, any residual antibodies from the first round will bind permanently to the membrane.

    User Guides

    Title
    Immobilon -P Blotting Sandwiches
    Immobilon-P Transfer Membrane User Guide
    Immobilon-Psq Transfer Membrane User Guide
    Immobilon®-FL Transfer Membrane User Guide