![]() Peroxide may be inactive, resulting in lower peroxidase signal Try lower concentration of blocking agentīuffers may contain sodium azide, which inactivates HRPĮCL detection reagents may be contaminated.Repeat using two membranes in case protein has transferred through the first membrane (over-transfer is especially likely with low-MW proteins).Optimize and check transfer conditions and setup (especially orientation to electrodes).Note how well any prestained molecular weight markers have transferred onto the blot.Confirm protein transfer by staining the membrane with Ponceau S and/or the gel with Coomassie Blue Dye, or use Bio-Rad’s Stain-Free Gels to verify transfer using our unique stain-free imaging technology.(see also Protein transfer or binding issues) (see also Signal Strength Problems > Faint bands, weak or no signal) A Method for Greater Reliability in Western Blot Loading Controls: Stain-Free Total Protein Quantitation Bulletin 6360.Trends in Protein Separation and Analysis - the Advance of Stain-Free Technology Bioradiations, Sept.Applications & Technologies: Stain-Free Imaging Technology.Try total protein normalization using stain-free technology instead of normalizing to a single housekeeping protein.If loading control expression varies with experimental conditions, try using another loading control Check that loading control expression is consistent across conditions using a secondary loading control.Loading control protein levels may vary between test and control conditions In addition to providing visual verification of transfer at every step, stain-free imaging enables total protein normalization in each lane, eliminating the need for housekeeping proteins as loading controls Visualize total protein on gels and blots using Bio-Rad’s Stain-Free Gels featuring our proprietary Stain-Free Technology. ![]()
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