Which is the best method to extract protein from cells in culture to do western blot analysis?
In particular I am working with cells from rat testes and they are very hard to homogenize in particular after some weeks of culture.
Not, I have not done the protein extraction yet.
It is the first time I work with this technique and I would like to do the lysis of cells directly on the plate to avoid cell loosing.
Do you know the recipe of a good lysis buffer?
After the lysis do I have to do the sonication in your opinion?
Thank you!
Alessandra
Lysis buffer:
20 mM Tris-HCl (pH 7,5),
150 mM NaCl,
1% Triton X-100,
50 mM NaF,
5 mM EDTA,
and a mixture of protease inhibitors (Roche Molecular Diagnostics, Meylan, France)
We use very similar protocol to Thomas, with little modification -
actually after washing plates with ice-cold PBS we immediately scrape cells, than transfer lysis buffer with cells in 1,5 ml epp tubes and incubate them for 10-20 min on ice - than start to centrifugate. All the next steps are the same.
It works.
Good Luck!
if you read GE healthcare protocol "Protein Sample Preparation" , you won't any trouble in work
http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/service-and-support/handbooks/
Dear Thomas,
sorry if I answer so late...
I think that there are not commercial mouse testis cell lines...Indeed, despite I work with testis tissue only since few time, I think is quite impossible to establish a cell line of whole testis cell population.
If you need to study your protein in this kind of tissue, I suggest you to take fresh tissue, dissociate it and put it in culture.
Dear all,
Do you have protocols for preparing the whole cell extract from frozen testis tissue?
Kind regards
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