3 replies to this topic

[MaggieRoara]

Hi everyone.
I am currently using the GST pull down technique to validate protein interactions between two of my proteins. I am expressing one protein in HEK293 and the other in ecoli cells. I am currently using frozen lysates. Will this affect my interaction studies?

[Penguin]

MaggieRoara, on Jul 6 2009, 03:31 AM, said:

Hi everyone.
I am currently using the GST pull down technique to validate protein interactions between two of my proteins. I am expressing one protein in HEK293 and the other in ecoli cells. I am currently using frozen lysates. Will this affect my interaction studies?

Did you put glycerol in your lysate before freezing? If not, ice crystals will have formed which may have denatured your proteins.  Also, do you have protease inhibitors in your lysate?

If yes to both these questions your pull down should work ok

P

[LucasRudy]

Penguin, on Jul 6 2009, 02:01 PM, said:

Did you put glycerol in your lysate before freezing? If not, ice crystals will have formed which may have denatured your proteins.  Also, do you have protease inhibitors in your lysate?

If yes to both these questions your pull down should work ok

P

Hi Penguin,
if you add glycerol to yr lysate before freezing, how do you get rid of the it ?? after you have thawn up sample and continue with the lysis

[Penguin]

LucasRudy, on Mar 13 2010, 12:59 PM, said:

Hi Penguin,
if you add glycerol to yr lysate before freezing, how do you get rid of the it ?? after you have thawn up sample and continue with the lysis

Hi,

The glycerol should not effect the pull down, up to around 10-20% anyway.  If you are worried about the glycerol you can always dialyse but you might lose some of your proteins and will dilute the sample down a bit.

P