Trouble concentrating cell extract -please help! - (Feb/22/2007 )

Hi out there,

I have run into a frustrating problem and I can't seem to find an answer to it. I am trying to concentrate a cell extract so that I can do Western blot for my proteins of interest. I am studying many samples, so we have essentially ruled out expensive columns and time-consuming dialysis. It seemed that precipitation was the answer for me. However, every time I try ppt an extract, it forms a little hockey puck that won't redissolve. I have try ppt'ing with ethanol and acetone - and stopped short of using TCA since I have heard that the pellets from that technique are even harder to redissolve. I have tried dissolving my pellets in Laemmli buffer and even 8M urea sample buffer while boiling or leaving them overnight but nothing is working! I even gave it a whirl with a little bit of DMSO. Would I have better luck with another technique like PEG ppt or Amm. Sulphate ppt? Is there something else to try?

Thanks so much,
Paul Kahlke

-kahlkep-

concentrating a sample does not necessarily mean precipitation; what about lyophilization to a highly concentrated but not dried form? one may work out a protocol to the tightest concentration without precipitates; starting sample of course should neither already contain precipitates nor should precipitate if cooled

-The Bearer-

QUOTE (The Bearer @ Feb 23 2007, 04:00 AM)

Thanks for the suggestion - that does sound like a good idea. However, I don't have access to lyophilization equipment. Is there another way to go about it?

-kahlkep-

QUOTE (kahlkep @ Feb 24 2007, 05:20 PM)

QUOTE (The Bearer @ Feb 23 2007, 04:00 AM)

Thanks for the suggestion - that does sound like a good idea. However, I don't have access to lyophilization equipment. Is there another way to go about it?

You can definately concentrate your protein via TCA precipitation. however, I will recommend you to lyse the cell with 1X laemmli buffer directly if you want the whole cell extract. In this way, you get much higher concentration. Of course, this is for western blot only, not for IP.

-Biomedmedia-

QUOTE (kahlkep @ Feb 25 2007, 02:20 AM)

QUOTE (The Bearer @ Feb 23 2007, 04:00 AM)

Thanks for the suggestion - that does sound like a good idea. However, I don't have access to lyophilization equipment. Is there another way to go about it?

-The Bearer-

the best way to concentrate would be a SpeedVac; may be you find one anyhow
alternatively, you may use an exsiccator with a vaccum pump to concentrate your probe; as there is no centrifugal force you have to be very careful;
[/quote]

Well! The SPEEDVAC system really help but most of all for volatile solvents ( best one for concentration HPLC probes with AcCN).

Exsiccator with Millipore vacuum membrane pump is time consuming but really available in labs. I think that you should use in this case protease inhibitors and ice

-circlepoint-

if you have chemists in your area, they often use rotavap.
these stuff can decrease the pressure at less than 76mBar.
At 76mBar and RT, the water goes to vapor phase, allowing you to concentrate your sample
but all these methds (rotavap, speedvac, lyophilisation) encounter a limit : salt concentration. I think you may not reasonably hope for more than 5X concentration.

-fred_33-

You could also try Millipore Microcon columns, which reduce volume by filtration through a dialysis membrane, retaining all of the large molecules. All you need is a centrifuge.

-phage434-

I have tried to solubulize protein in 8M guanidine HCl in 10 mM Tris (pH8) and then dialyzed against 6MUrea/2M thiourea.
Or you can first try to see your protein is dissolved in 6M urea/2M thiourea.
This method saved me when I was struggling to dissolve oyster shell matrix protein for 2D gel back in late 80's.
Good luck!

-kawaguchi2001-

QUOTE (kahlkep @ Feb 22 2007, 08:06 PM)

Try to sonicate probes and them take aliquote add sample buffer and do western. WB is sensitive technique and I think you should see qualitative your interesting protein. Or you have another task?

-circlepoint-