Protein Extraction

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Uzma Faridi
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Protein Extraction

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Hi,
I want to isolate protein from cancer cell lines.
i had tried several methods but i am not getting sufficient amount of protein as i have to perform western blotting.
Kindly suggest the appropriate method for that.

Thanks in advance
Regards
Uzma

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Hi,

Hi,
I have a couple of questions:
Do you any idea about the protein yield .
Do you use whole cell extract or supernetant or purified protein for performing western blotting.

Uzma Faridi
Uzma   Faridi's picture
 Hi,

 Hi,
Thanks a lot for replying :)
i m using whole cell extract n the protein is of cytoskeleton ..

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Hi,

Hi,
As we all know  for the western blotting need very minimum amount of protein (pg-ng). Probably you can start with more amount of cells,at the same time  make sure that cell lysis is complete( for making the whole cell extract ).If posssible collect the different time interval samples of Cancer cell line.

All the best

Balu..

g a
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Hi Uzma

Hi Uzma

There may be several reasons for not getting enough protein fo western blotting.

1. Insufficient starting materials as cells.
If you are using 24 well plates then in our lab we pool cells from 6 wells of 24 wells for getting sufficient material for 2-3 PAGES.
2. Lysis / Extraction buffer not working properly. Check the composition and references for the optimal buffer.
3. Protease inhibitors: If you are not extracting yr proteins with protease inhibitors there is a fair chance that the proteins get degraded.

For western blotting Anywhere between 10 - 40 micrograms protein load is sufficient but one must never forget the abundance of the protein in question is very important point for consideration and one may be required to load higher amounts for very low copy number proteins.