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why need to do protein assay before western blot? - (Mar/11/2008 )

Anybody could tell me the reason why we need to do protein assay before western blot?

I know protein assay is for protein quantification, if i don't do protein assay what will be the disadvantage for western blot?

thx

-coolku-

Hi.
I am doing western quite a lot, the only thing I do after bloting is a pounce staining to the membrane to check the amount of protein extracted and then detection. before blotting i don't do anything...... mellow.gif

-solmaniar-

QUOTE (coolku @ Mar 11 2008, 10:52 AM)
Anybody could tell me the reason why we need to do protein assay before western blot?

I know protein assay is for protein quantification, if i don't do protein assay what will be the disadvantage for western blot?

thx


1. you won't know how much total protein you are putting in each well and this could lead you to overload your gel.
2. If you want to determine relative differences in protein levels between different samples, then you should try to load equivalent amounts of total protein. You can always use a control such as actin, but this makes it easier in the long run and your comparative blots will look neater.
3. You can do the western without protein quantification first. There are several protocols where cells are lysed directly into loading buffer and therefore quantification is not applicable, but if you do this then you should still try to have some idea of how much relative protein you are loading for each sample.

-smu2-

QUOTE (solmaniar @ Mar 11 2008, 08:33 PM)
Hi.
I am doing western quite a lot, the only thing I do after bloting is a pounce staining to the membrane to check the amount of protein extracted and then detection. before blotting i don't do anything...... mellow.gif


thanks


QUOTE (smu2 @ Mar 11 2008, 08:46 PM)
QUOTE (coolku @ Mar 11 2008, 10:52 AM)
Anybody could tell me the reason why we need to do protein assay before western blot?

I know protein assay is for protein quantification, if i don't do protein assay what will be the disadvantage for western blot?

thx


1. you won't know how much total protein you are putting in each well and this could lead you to overload your gel.
2. If you want to determine relative differences in protein levels between different samples, then you should try to load equivalent amounts of total protein. You can always use a control such as actin, but this makes it easier in the long run and your comparative blots will look neater.
3. You can do the western without protein quantification first. There are several protocols where cells are lysed directly into loading buffer and therefore quantification is not applicable, but if you do this then you should still try to have some idea of how much relative protein you are loading for each sample.


thanks! it's helpful!

-coolku-