I am using BSA for blocking membrane for Phosphospecific antibodies but getting much background, could any one help me how to overcome this problem?
Dear Yogendra -- as Oliver suggested, milk is usually a better blocker for non-specific binding than BSA and most other proteins. See also the comment by Erin Stephenson at the attached RG link. Many years ago, we found that non-fat dry milk (eg Carnation) rehydrated to 5% was a more effective pre-treatment blocker for microtiter plate ELISAs than BSA and most other proteins (file attached). Casein and high-density lipoprotein (HDL) were the only pre-treatment proteins as effective as rehydrated milk. When used in the solution matrix during the binding incubation, rehydrated milk and gelatin were the most effective.
https://www.researchgate.net/post/How_to_get_rid_of_high_background_non-specific_negative_ghost_bands_on_a_western_blot_of_a_tissue_homogenate
thanks i will use tomorrow skim milk blocking agent for phosphospecific kinase protein and see what will be ... because previously I was not used milk buffer because they contain phosphate but in skim milk free phosphate.
if i will get result then again contact with you..
Dear Yogendra -- as Oliver suggested, milk is usually a better blocker for non-specific binding than BSA and most other proteins. See also the comment by Erin Stephenson at the attached RG link. Many years ago, we found that non-fat dry milk (eg Carnation) rehydrated to 5% was a more effective pre-treatment blocker for microtiter plate ELISAs than BSA and most other proteins (file attached). Casein and high-density lipoprotein (HDL) were the only pre-treatment proteins as effective as rehydrated milk. When used in the solution matrix during the binding incubation, rehydrated milk and gelatin were the most effective.
https://www.researchgate.net/post/How_to_get_rid_of_high_background_non-specific_negative_ghost_bands_on_a_western_blot_of_a_tissue_homogenate
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