Technique / Proteomics and protein biochemistry / Western blot assay
Western-Blot: PBS/TBS
Western-Blot: PBS/TBS (archive)
Does anybody know if it makes a difference for certain antibodys (for
protein-detection on a western-blot) to use PBS instead of TBS as basic for
all the incubation- and washing-buffers?
The reason why I´m asking is the following one:
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Last update 08-Mar-2002, Rating Good of 1 votes.
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hi, james.
the reason i think your prof wants you to do the Western anyway is because there could be MANY proteins that have the same or similar molecular weight as the one you are looking for.
the ponceau staining only lets you know if you transfered the protein succesfully to the membrane, but it does not tell you the position or quantity of you goal protein.
you could actually be staining a bunch of look-a-like-proteins.
i think antibodies are the most specific little gadgets on earth.
use them wisely.
cárol
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Hi,
Having phosphate ions in washing buffer may be somehow inhibitory to activity of few enzymes(linked to secondary antibody) such as alkaline phosphatase.i such cases it is strongly recommended to use a buffer such as TBSwhich is free of these ions.for few other enzymes such as HRP both buffers seem to work equally well.
neetu
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hi,
I have just started my Masters program and i have one qn. I dont know how worth is that. I stained the membrane (nitro cellulose) with the poncaeu and my prof told me that this is the protein am looking for. then she asked me to do the western, i could not understand that point. whats the prupose of doing the western once v got the band in the poncaeu.(am sure the antibody is specific to the protein for which am looking for). Is there is anyone to guide me?
james
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