While detecting the phospho-protein people block their NC or PVDF membranes with BSA (3-5%) rather than NFM. Can any one please explain why BSA blocking is preferred for detection of phosphorylated form of a protein?
Milk contains casein which is a phospho-protein. Casein causes high
background because the phospho-specific antibody detects the casein present in the milk.
Milk contains casein which is a phospho-protein. Casein causes high
background because the phospho-specific antibody detects the casein present in the milk.
Vanessa Rivera-Amill Thanx.....Understandable.....But apart from casein, is there any other possibility like the presence of phosphatases in NFM that may effect the phosphorylation status of phospho-protein?
I understand cell/tissue homogenates do have phosphatases that's why we use inhibitors to overcome their enzymatic activities. But I think blocking the membranes with Non-Fat Milk will not have any dephosphorylating effect on phosphor-protein because the proteins on membranes are already denatured (as we add SDS and also heat our protein samples during preparation) and thus are not labile to be dephosphorylated by phosphatases (if any in non-fat milk). Any ways casein seems to be the predominant factor effecting the detection of phopsho-protein on membranes. Thanx again for information/help.
Although Vanessa answered your question very well; however milk also contain traces of casein. I would like to add that, as alternative for phospho-proteins, you always can pre-block the membrane with normal sera from the same host-specie used to produce the 2nd Ab, use BSA (protease/IgG free) or try protein-free blocking buffers.
Thanx Lirola.....I got the main point...but have little confusion.....Why to use protease free BSA? Because I believe the protein samples that we load on PAGE are already denatured and should not be lysed by proteases?
The key here is that non-fat milk contains tyrosine phosphorylated proteins, predominately, which can increase the background from your primary antibody (nothing to do with the denatured proteins on the blot) and sometimes occurs irrespective of whether your antibody is anti-serine/threonine targeted....
Dear Muhammad,
As you point out very well, protease-free BSA may not have too much sense is this special case; however, the one I usually use is also IgG free which may became an antigen for cross-reacting secondary antibodies in your WB. That second property might be important for you as the result of these interactions may be loss of desired antibody activity, loss of antibody stability, and/or increased background. When I compared that product against other BSA commercial preparations (even with the highest levels of purity) I found extraordinary differences.
@Lirola...Agree....IgG can interfere and IgG free BSA might be the best choice for blocking the membranes, while detecting p-proteins....Thanx again.....
@Richard...The presence of other tyrosine phosphorylated proteins (in addition to p-casein) seems to be the additional point, explaining why BSA should be preferred over NFM for p-protein detection. Thanx for information.......
BSA is the best blocking agent to reduce the background, since it contains mainly albumin compared to NFM, which has other p-proteins. lalitha kabilan
Hi @Lirola and @ Imran - thanks for the tip about IgG free BSA, will definitely give that a go!
Thanx Lalitha....Things are getting more and more clear......
@Richard....I know every one deserves to be thanked and appreciated for sharing their knowledge here in the thread....but the credit for IgG related information goes to Lirola............
Milk-based blockers may contain IgG that can cross-react with anti-goat or anti-sheep antibodies. This increases background and can reduce antibody titer. Also, high concentrations of milk blocker can cause loss of blotted proteins from the membrane (J Immunol Methods 122 (1):129-35 (1989)).
Thanks Amy Schutz........So the bottle neck is IgG and also the phospho-proteins present in NFM......and that's why BSA is a preferred choice for blocking......
Hi Mats....Thanx for explaination... Plz refer to the following link, you will find similar discussion there...
https://www.researchgate.net/post/Western_blotting-using_BSA_or_milk_And_TBS-T_or_PBS-T11?exp_tc=tprc
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