Immobilon® Membranes, Sandwiches and Blotting Filter Paper
Immobilon® PVDF membranes are the ideal transfer membranes for protein blotting applications.
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Documentation
References
| Reference overview | Application |
|---|---|
| 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) |
| 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 | |
| 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 |
| 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 |
| The clinical usefulness of the measurement of cytokines. Bienvenu J et al.,Clin Chem Lab Med. 2000 Apr;38(4):267-85. Review. Clin Chem Lab Med. 2000 Apr;38(4):267-85. Review. 2000 | ELISPOT Assays |
| Amino Acid Analysis: A comparative Study of stains on PVDF membranes ,Journal of Biomolecular Techniques, Vol II, Issue 1, March 2000. Journal of Biomolecular Techniques, Vol II, Issue 1, March 2000. 2000 |
FAQ
| Question | Answer |
|---|---|
| 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. |
| 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. |
| 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. |
| 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. |
| 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. |
| 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. |
| 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). |
| 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. |
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