How to dissolve TCA/acetone precipitated proteins? (View forum version)



Bacillus_Guy

Posted 30 July 2013 - 01:17 AM

Hey everybody,

 

how should I dissolve TCA or acetone precipitated proteins other than with Laemmli buffer?

 

Should I use different buffers for whole cell extracts and membrane proteins?

 

I'm asking, because I want to determine the protein amounts with a Nanodrop before loading the samples on a SDS gel and as far as I know it's not possible with the loading buffer containg bromphenol blue. right?

 

Thanks in advance,

Bacillus_Guy

 


mdfenko

Posted 30 July 2013 - 04:01 AM

you can prepare loading buffer without bromphenol blue (bpb) and add some after determining protein. if you want to spend a little money then there is an ir spectrometer (direct detect) available from millipore which will be unaffected by the bpb.

 

you can wash residual tca out of the pellet with acetone or ether.

 

if you need to neutralize remaining acid (you'll know if it is required when you add the bpb if it turns yellow), you can use a few ul of 0.5 or 1.0M tris (we use 0.5M pH 9, you can also use tris base) until it just turns blue.