i read somewhere if i know the concentration of each primary antibody of my interest i can just incubate all the PVDF membrane with a solution containing all the antibodies in specific concentration...is it true and applicable practically?
If the antibodies are highly specific and proteins of interest are well resolved and not overlapping then yes, an antibody cocktail can be used.
I agree with Syed.
Be careful with the molecular weights of target proteins, and chosse the right acrlylamide concentration.
This honestly sounds to me like a recipe for disaster. If you intend to quantify relative expression levels of proteins (and this should always be your highest priority), I would strongly recommend not to add more than one antibody onto your PVDF membrane because of the following reasons: Firstly, what determines your signal intensity of your major (and minor unspecific) band(s) is the affinity of the antibody and all your antibodies will have affinities that differ by orders of magnitude (happy diluting). To titrate your antibody stocks so that your signal intensities match will definitely take you longer (if it works in the first place) than developing two or three PVDF membranes. Secondly, you will have realised that for some antibodies your chemiluminescence signal is “burnt” within a minute, whereas for other antibodies you will have to expose your film for 5-10 minutes. For meaningful expression results your signal should NOT be saturated (i.e. black), so you are facing a serious timing problem of you have 2/3 antibodies on the same membrane. Should you manage to find an antibody combination where your trick works, don’t try it with a different combination, as it will fail.
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