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.To verify protein transfer, stain the membrane with Ponceau S after blotting.To determine if there is residual, untransferred protein remaining on the gel, use a total protein stain on the gel after transfer.For example, Coomassie and colloidal gold are not compatible with downstream steps (see Bio-Rad Protein Stains and the Protein Stain Selection Guide) If planning to use the blot in downstream steps, make sure that your stain can be removed or is compatible with antibody detection. To determine whether the changes in loading control levels are due to differences in the amount of sample loaded, or if the differences are caused by variations in expression of the loading control proteins, use total protein stains (e.g., Ponceau S, Coomassie, colloidal gold, or SYPRO Ruby) to visualize proteins on gels and blots before and after transfer to determine relative protein loading.Check concentration of protein samples (e.g., using Bradford or Lowry protein assays).Initial sample quantitation (O.D., weight, cell count, etc.).Check that total protein levels are consistent:.Samples may have different amounts of total protein Variation observed among the loading controls in each lane
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