![biorad western blot protein ladder biorad western blot protein ladder](https://www.biolegend.com/Files/Images/BioLegend/westernblot/PrimeStep_Protein_Ladder.png)
Include a positive control in experiment (all PrecisionAb Antibodies include a positive control lysate).For more information, see Tips for Caring for Your Antibody.If storing an antibody for a very long period of time, store at –80✬.Use fresh aliquots of antibody that have been stored at –20✬ or below.Freeze aliquots of antibody and only thaw one at a time as needed for blots store thawed aliquots at 4☌.Test on a dot blot at several concentrations.Validate target and antibody combinations using checkerboard screening protocolsīio-Rad now offers PrecisionAb™ Validated Western Blotting Antibodies for superior performance in western blot detection.Īntibody activity loss due to long-term or improper storage.
![biorad western blot protein ladder biorad western blot protein ladder](https://www.selectscience.net/images/articles/7380_BioRad_ChemiDocTouch_140423_1895.jpg)
Check datasheet for recommended conditions.Bio-Rad now offers high-quality antibodies for all applicationsĪntibody not suitable for western blotting Test and optimize antibody on dot blots.Lower temperature, reduce detergent concentration, reduce ionic strength.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.
![biorad western blot protein ladder biorad western blot protein ladder](https://www.mdpi.com/ijms/ijms-18-00089/article_deploy/html/images/ijms-18-00089-g004.png)
(see also Protein transfer or binding issues) (see also Signal Strength Problems > Faint bands, weak or no signal)