TCA precipitation - (Jul/29/2008 )
Hi
I am hoping to try a TCA precipitation as I want to concentrate my protein samples for western blotting.
The protocol I will be using is as follows:
-add an equal volume of 20% TCA to protein (i will have about 150ul of protein, at a concentration of about 2ug/ul)
-incubate for 30mins on ice
-spin at 5000rpm for 15mins
-remove supernatant
-add approx 30ul cold acetone & spin for 5mins at 4degrees
-remove supernatant & dry pellet
-resuspend samples in SDS PAGE loading buffer. Load to SDS PAGE after heating at 65degrees for 3mins
I just have a couple of questions about this protocol that I was hoping someone could help me with.
Firsty, is it likely that i will see a pellet at any stage, considering the amount of protein that I have.
Secondly, is it possible to freeze my pellet at -20degrees before resuspending in the loading buffer or would I have to resuspend immediately?
Thanks for any advice:)
Firsty, is it likely that i will see a pellet at any stage, considering the amount of protein that I have.
-Yes, for 300 ug of protein.
Secondly, is it possible to freeze my pellet at -20degrees before resuspending in the loading buffer or would I have to resuspend immediately?
-Yes you can freeze it.
Two comments.
I would spin at top speed (13 000 rpm) in a benchtop centrifuge.
Wash with a bigger volume of acetone to make sure you remove all trace of TCA - if there's some acid left your loading buffer will turn yellow.
-Yes, for 300 ug of protein.
Secondly, is it possible to freeze my pellet at -20degrees before resuspending in the loading buffer or would I have to resuspend immediately?
-Yes you can freeze it.
Two comments.
I would spin at top speed (13 000 rpm) in a benchtop centrifuge.
Wash with a bigger volume of acetone to make sure you remove all trace of TCA - if there's some acid left your loading buffer will turn yellow.
Great, thanks a million:) And thanks for the extra comments-they make sense so I think I'll alter my original protocol.
-Yes, for 300 ug of protein.
Secondly, is it possible to freeze my pellet at -20degrees before resuspending in the loading buffer or would I have to resuspend immediately?
-Yes you can freeze it.
Two comments.
I would spin at top speed (13 000 rpm) in a benchtop centrifuge.
Wash with a bigger volume of acetone to make sure you remove all trace of TCA - if there's some acid left your loading buffer will turn yellow.
I usually make up some basic (pH 9 or so) SDS-loading buffer for running TCA precipitation. If there is any residual TCA left it will be neutralized by the basic buffer and will not turn yellow.