far western - Would the expression of the protein in mammalian cells be enough for far wester (Sep/01/2006 )

Hi everybody!!!

I'm new in this field and therefore any reply would be appreciated a lot.
I need to confirm the interaction of 2 proteins to show that it's a direct binding.
And I want to do Far western, so I need a pure protein.
I wonder is it possible not to go to Ecoli system and to do it in mammalian cells,
since I've got an expression vector with protein of interest with flag tag.
If I overexpress the protein in mammalian cells and than purify with anti flag column would the protein be enough for far western???
i don't now how good the flag tag is for this purpose. Has anybody purified the protein with flag tag????
Is his tag better for this purpose in case if I have to clone the gene of interest in the E.coli expression vector??
Thank you in advance,

Karmen

hi
i'm currently dealing with flag tagged proteins. I've transfected HeLa cells (and for your convenience you may use Hela cells in suspension with joklik medium), and made a selection by antibiotic resistance. The ORF is under control of the CMV promoter.
I've extracted half of a 15cm plate and got a good expression and purfication.
I've checked the expression with 20µg wblot, and the result was a nice band.
The purification is ok also.
For far western, if i needed to do that, i would do 4-5 plates and extraction/purification of the protein.
The accessibility of the flag tag may e tough, as the fact of detaching protein partners of your protein. you may so increase the salt concentration.
I've done purifications on several plates, and the FT of the first column is reusable on an other one and gave me roughly same activity as the proteins recovered in first column.

Fred, many thanks for your kind reply.
I found out someone in the lab has got another GST tagged plasmid.
So, I will try to express in coli and than let's see what comes out.

I am gonna clone another gene in mammalian expression vector, could anybody advise me what kind od tag is better to take which would work good for both IP or Western blot.
Greetings,

Karmen