Protein Precipitation (wessel and Fluege method) - 2 questions (Aug/09/2006 )

I met some problem with my protein precipitation.

1, In my protocol, the pH of chloroform should be 6, but when I opened a new bottle, it is about 4.5, my question is: can I ajust pH of chloroform by adding NaOH?

2, by using this chloroform, and following the protocol, I got something white attached to my eppendorf tube, but it seems quite hard to be disolved in Sample Buffer for SDS-PAGE, I was wondering, is that real protein? why does it become insoluable....

The way to measure the pH of chlroroform is to wash /saturate it with water and measure the pH oif water after phase separation. I dont see a need to adjust pH for CHCl3.

Proteins tend to denature and aggregate at the water/organic solvent interface. It may take longer to re-dissolve or it may not be soluble at all. If you dont have your sample fully re-dissolved you may lose some protein bands. You can run a gel with sample lysed with SDS sample buffer alone as positive control to see if your interested band(s) are present in the soluble fraction. There are so many ways to prepare your sample. I am sure there is alternative for that.

Thanks,
I will try your way... actually, i just drop some cloroform to measure the pH....

and I just run another gel, tomorrow I will know whether that white powder is protein or not..(with total lysate as positive control )

I got very bad results.....

The white powder I saw was not protein at all......... I did a control experiment with elution buffer alone. After precipitation, this white powder appeared...

The chloroform dissolved parts of your tube. the substance is plastic. chloroform is a very volatile solvent, and despite what any company will say about the quality of their products (any plastic tube), they will all dissolve.

I met some problem with my protein precipitation.

1, In my protocol, the pH of chloroform should be 6, but when I opened a new bottle, it is about 4.5, my question is: can I ajust pH of chloroform by adding NaOH?

2, by using this chloroform, and following the protocol, I got something white attached to my eppendorf tube, but it seems quite hard to be disolved in Sample Buffer for SDS-PAGE, I was wondering, is that real protein? why does it become insoluable....

then could someone tell me what is the solution to this problem. we also had this white precipitate problem for methanol-chloroform precipitation... one cannot use this protocol then or?

is there any alternative method for precipitation prior to In-Solution Tryptic digestion. I would prefer not to use TCA precipitation as one may not be able to get rid of the acid and this may not be so good for subsequent tryptic digestion (in-solution)

thank you for helping.

regards
rajgene