PROTEIN PURIFICATION WOES - AN UPDATE!!!

[Andy Hill]

Hi Again

Firstly, thanks to all those who e-mailed me and posted messages. There
were several suggestions which I am following up, namely:

1) Reducing pH to <4 to ppt DNA (or using PEI, protamine sulfate, etc...)

2) Trying Exclusion on Q-media first then running through S-media

3) Dilution of detergent to levels below CMC

4) Alternative buffer systems

5) Checking pI of the protein

(sorry if I excluded any others, but I took them all into consideration!)

OK, I tried diluting out the detergent to below CMC and running on
S-Sepharose, but still got flat line after the void on the chart. I also
tried reducing the pH to below 4 but this didn't seem to work either. I
couldn't try the precipitations because we didnt have any PEI or protamine
sulfate at the time!

However, I did manage to get a good separation of proteins by running my
cell extract on a Q-column (I used a Bio-Rad Econo-Q-Column). So I now
have separated my protein away from a lot of other acidic proteins. I am
still analysing the fractions but I guess the next step would be to put
the excluded fractions on the S-Sepharose. Not bad for a Friday
afternoon!!!

I have just looked at the predicted net charge of the protein at pH 7 and
it is +7 - is this enough for good separation? It goes up to +28 at pH 4
but I would like to avoid acid treatment on the protein.

Anyway, thanks again for your ideas!

Andy