Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. YALOW RS, GLICK SM, ROTH J, BERSON SA (1964) Radioimmunoassay of human plasma ACTH. Sarge KD, Park-Sarge O-K (2009) Detection of proteins sumoylated in vivo and in vitro. Biochim Biophys Acta, Mol Cell Res 1833:812–822 Voelkel T, Andresen C, Unger A et al (2013) Lysine methyltransferase Smyd2 regulates Hsp90-mediated protection of the sarcomeric titin springs and cardiac function. Pere-Brissaud A, Blanchet X, Delourme D et al (2015) Expression of SERPINA3s in cattle: focus on bovSERPINA3-7 reveals specific involvement in skeletal muscle. Proc Natl Acad Sci U S A 79:5249–5253Ĭonte A, Sigismund S (2017) Methods to investigate EGFR ubiquitination. Put them into chemiluminescent imager forīefore proceeding Western Immunoblotting, add specific Blocking Peptide (Refer to blocking peptide catalog#) to the diluted primary antibody in a molar ratio of 10:1 (peptide to antibody) and incubate the mixture at 4℃ for overnight or at 37 ☌ for 2 hours.Īfter blocking membrane,wash membrane with TBST for 1-2 min, then add phosphatase to the diluted primary antibodywith the appropriate concentrationand incubate the mixture at 4℃ for overnight or at 37 ☌ for 2 hours.Wu WC, Walaas SI, Nairn AC, Greengard P (1982) Calcium/phospholipid regulates phosphorylation of a Mr “87k” substrate protein in brain synaptosomes. Chemiluminescent substrate lay evenly over the membrane. Prepare and use the Chemiluminescent substrate according to the manufacturer’s instructions.Ģ. Wash the membrane 3 times for 5 minutes each time at room temperature inġ. Instructions) in Blocking Solution for 1 hour at room temperature with shaking.Ģ. Incubate membrane with diluted HRP-conjugate secondary antibody (according to manufacturer’s Incubate the membrane with diluted primary antibody for 2 hours at 37 ☌, or overnight at 4 ☌ withģ. Dilute primary antibody at the appropriate dilution in Blocking Solution.Ģ. Block membrane by incubating 1 hour at room temperature or overnight at 4 ☌ with shaking in Blocking Run the gel and transfer to nitrocellulose for western immunoblotting.ġ. Load equal amounts (30–45μg) cell lysate onto SDS-PAGE gels using loading tips, along with molecularĨ. Centrifuge at 14,000 rpm in a microcentrifuge for 5 min.ħ. Boil each cell lysate at 100 ☌ for 5 min.Ħ. To the remaining volume of cell lysate, add half volume of 5X SDS Sample Buffer.ĥ. To perform a protein assay, determine the protein concentration for eachĤ. Detach adherent cells using a cell scraper or centrifuge suspended cells, and resuspend in 1X Cellģ. Treat cells by specific regulator for desired time, wash cells with 1X PBS.Ģ. Westerns are performed using cell lysates from harvested cells.ġ. Blocking Buffer:5% nonfat milk, 1X TBS, 0.1% Tween-20ħ. 10X Tris Buffered Saline (TBS):To prepare 1 L of 10X TBS: 24.2 g Tris base, 80 g NaCl adjust pH to 7.6Ħ. Transfer Buffer:25 mMTris base, 0.2 M glycine, 20% (or 10%)methanol (pH 8.5)ĥ. 5X SDS Sample Buffer:1M Tris-HCl (pH 6.8), 10% SDS, 50% glycerol, 500mM DTT, 0.05% BromophenolĤ. 1X Cell Lysis Buffer with protease/phosphatase inhibitorsģ. 10X Phosphate Buffered Saline (PBS): To prepare 1 L of 10X PBS buffer: 80g NaCl, 2g KCl, 36.3g
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