Acquire images using darkroom development techniques for chemiluminescence, or normal image scanning methods for colorimetric detection. Depending on the primary antibody, is sometimes possible to detect also the protein ladder, this does not means that your western blot is wrong or that the bands that you see are unspecific.Remove excess reagent and cover the membrane in transparent plastic wrap. For signal development, follow the kit manufacturer’s recommendations.Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer at room temperature for 1 h on a shaker with a low setting.Wash the membrane in three washes of TBST, 10 min each.Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer for 1 hour at room temperature on a shaker with a low setting.Wash the membrane with 1x TBST for 10 minutes.Block the membrane for 1 h at room temperature or overnight at 4☌ using 5 % blocking buffer.Once complete, wash twice for 10 minutes in deionized water before drying and storing at 4☌ or continuing with antibody staining.Each protein can be stained with a different color for easy identification or labeled with affinity tags for western blot detection. Complete a wet transfer at 500 mA, for 1h, at 4☌ using the pre-chilled transfer buffer. Recombinant protein ladders are engineered to produce tight bands, evenly spaced molecular weights, and other specific traits.Familiarize yourself with the protocol and check the common pitfalls. If all the bands on your blot including the molecular weight ladder are difficult to see, it could indicate a problem with your technique rather than the protein you’re trying to detect. Scrape adherent cells off the dish using a cold plastic cell scraper, then gently. All bands, including the ladder, are faint or have no signal. Aspirate the PBS, then add ice-cold lysis buffer (1 mL per 10 7 cells/100 mm dish/150 cm 2 flask 0.5 mL per 5x10 6 cells/60 mm dish/75 cm 2 flask). Immerse PVDF membrane, filter paper, and sponge in 1× transfer buffer for 30 min before transfer. Place the cell culture dish on ice and wash the cells with ice-cold PBS.Activate the PVDF membrane with 99.5% methanol for 15 seconds.Immerse the gel in 1× transfer buffer for 40 min.Transferring the gel from the plate to the membrane A reducing gel should be used unless non-reducing conditions are recommended on the antibody datasheet. We recommend following the manufacturer’s instructions. The time and voltage may require optimization. Run the gel for ~1.5 h at 150 V, referring to the molecular weight markers and using the pre-chilled running buffer.Use fresh aliquot of antibody that has been stored at -20☌ or below. Test on a dot blot at several concentrations. Antibody has lost activity due to long term or improper storage. Check datasheet for recommended conditions. Load equal amounts of protein into the wells of the SDS-PAGE gel, along with the molecular weight marker. Antibody not suitable for Western blotting.MacPhee DJ (2010) Methodological considerations for improving Western blot analysis.Dissolve components in 3.5 L of water initially, then make up to 5 Lĭissolve components in 3.5 L of water initially, then make up to 10 LĪdd 10 % methanol to transfer buffer just before useĭissolve in deionized water, adjust pH to 7.4 using HCl, then make it up to 22.5 L with deionized water.Īdd 2.25 L 10X TBS and 22.5 mL Tween-20, make up to 22.5 L with deionized water Biotechniques 11:14–16ĭubitsky A, DeCollibus D, Ortolano GA (2002) Sensitive fluorescent detection of protein on nylon membranes. Sandhu GS, Eckloff BW, Kline BC (1991) Chemiluminescent substrates increase sensitivity of antigen detection in western blots. Here are our recommendations: Mini-gel: 5 L per well (0.751.0 mm thick) or 10 L per well (1.5 mm thick) Large gel: 10 L per well (0.751.0 mm thick) or 20 L per well (1.5 mm thick) Incomplete or poor transfer. Load an appropriate volume of the ladder onto the gel. Methods Mol Biol 45:115–127Īlegria-Schaffer A, Lodge A, Vattem K (2009) Performing and optimizing Western blots with an emphasis on chemiluminescent detection. Not enough volume of ladder loaded on the gel. Methods 38:283–293įowler SJ (1995) Use of monoclonal antibodies for western blotting with enhanced chemiluminescent detection. 30.3 g Tris ( 25 mM Tris in 1x running buffer) 144 g glycine ( 192 mM glycine in 1x running buffer) 10 g SDS ( 0.1 SDS in 1x running buffer) o The pH of the buffer should be 8.3 if carefully. Kurien BT, Scofield RH (2006) Western blotting. Walker JM (1994) The bicinchoninic acid (BCA) assay for protein quantitation. Methods Mol Biol 424:139–146įriedenauer S, Berlet HH (1989) Sensitivity and variability of the Bradford protein assay in the presence of detergents. Methods Mol Biol 424:35–42Ĭordwell SJ (2008) Sequential extraction of proteins by chemical reagents. Weiss W, Görg A (2008) Sample solubilization buffers for two-dimensional electrophoresis. Mahmood T, Yang PC (2012) Western blot: technique, theory, and trouble shooting.
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