Biorad western blot protein ladder trial#

Increase the antibody concentration 2–4 times higher than initial trial.Wrong concentration of antibody or low affinity to the target protein Repeat experiment with the correct antibody combination.Reprobe with correct secondary or strip blot and reprobe if necessary.Retrace steps to check compatibility between primary and secondary antibodies.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

Biorad western blot protein ladder verification#

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