![]() ChemiDoc MP Imaging System, 30 sec ( ) film, 30 sec ( ). Other techniques including immunohistochemistry and immunogold can further evaluate the levels of the various antioxidant enzymes in tissues and cells. Antibody-HRP conjugates are superior to antibody-AP conjugates with respect to the specific activities of both the enzyme and antibody. The iBright Imaging Systems streamline the imaging experience with a combination of powerful hardware, automated features, and an easy to use interface. The manual process is not only laborious, but also risks introducing procedural variations from fluid delivery, washing, and timing. The generally low quality of western blotting data has led scientists to question whether we can trust protein quantification when using western blots. in 1979 and is now a routine technique for protein analysis. HRP functions optimally at a near-neutral pH and can be inhibited by cyanides, sulfides and azides. ![]() If the film is not exposed long enough (underexposed), the signal will not be visible. "House-keeping proteins should not be used for normalization without evidence that experimental manipulations do not affect their expression"*, Solution: Use Total Protein Normalization (TPN) instead of house-keeping proteins, "Methods including detection of enhanced chemiluminescence using X-ray film have a very limited dynamic range"*, Solution: Use a digital imaging system with up to 4 logs of dynamic range, "A description of the data supporting the specificity of all antibodies is required"*, * Revised guidelines for authors from The Journal of Biological Chemistry. Observe the developed film and put another film covering the blotting membrane if needed.Western blotting is a powerful technique that enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membranes.Develop the film by using a developed machine. Cover the membrane with a chemiluminescence film.Dry the blotting membrane with a filter paper to eliminate excess of ECL.Develop the blotting membrane with 5 ml of ECL for 1 min.Wash the blotting membrane four times with PBS + 0.1% Tween 20 for 15 min each.Incubate with the corresponding secondary antibody diluted 1:2000 for 1 hour at room temperature.Wash the blotting membrane three times with PBS + 0.1% Tween 20 for 15 min each.Use another primary antibody as loading control (tubulin, GAPDH, lamin, etc). Incubate the blotting membrane with the primary antibody for 1 hour at room temperature.Incubate the membrane in PBS + 2.5% dry milk powder overnight at 4º or for two hours at 37º to block non-specific binding to the membrane.Fill the tank with the blotting buffer ( Appendix II) and connect it to the powerPac Power supply 2 hours at 200 mA (or overnight at 20mA).Put it in the electrophoresis blotting module. Remove the air bubbles by gently rolling a Pasteur pipette over the pad. Prepare the blotting “sandwich” using the gel holding cassette.Cut and wash the blotting membrane in methanol and in blotting buffer for 10 min.Wash the gel in blotting buffer ( Appendix II) 10 min.Connect the tank to the power Pac Power supply and run the gel at 100 V until the gel front reaches the bottom.Use one well to load a molecular weight standard.Centrifuge the samples and heat for 5 min at 95º, centrifuge again and load into the gel.Fill the tank with the electrophoresis running buffer ( Appendix II).Prepare lysates by mixing with loading buffer and load as follows (in case of transfected cells put between 10-30ug/well, in case of no transfected cells or tissue extracts put 200ug/well).Prepare the stacking gel and put the comb inside it. Prepare the running gel percentage according to the expected molecular weight of the antigen ( Appendix I).ECL detection system (GE Healthcare RPN2109).Conjugated secondary antibody (DAKO polyclonal anti mouse/rat/rabbit HRP).30% acrylamide-bisacrylamide solution (Bio-Rad 161-0158).Nitrocellulose membrane (Millipore IPVH00010).Home > Protocols > Western Blotting (WB) protocol Western Blotting (WB) Protocol created by Lorena Maestre - Monoclonal Antibodies Unit, Centro Nacional de Investigaciones Oncológicas Material
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