concentration of protein in urea - I need help to concentrate proteins (by precipitation?) that are in 8M (Feb/06/2007 )
Hello all,
In my research I perform protein purification on columns of various types in denaturing conditions - 8M urea. As you all know many times the fractions are too dilute regarding the protein so they must be concentrated before running on SDS-Page.
My problem is as follows: I need to concentrate the proteins which are in 8M urea. I don't care what happens to the protein because I need it only for SDS-Page analysis.
So far I've tried several methods (TCA precipitation, Acetone precipitation and more) but non of them worked. The only method that works is ultrafiltration like centricons but it costs too much.
Please advise,
Joshua
use ultrafiltration anyway. you can clean and reuse centricon filters if you store them right. or switch to stirred cells. the initial outlay is greater but the membranes can be reused until you damage them.
First I want to thank you for replying.
Second, using amicon may present a problem because I have many samples that I want to examine simultaneously on SDS-Page. I only have one amicon device to concentrate the samples.
What about UPPA reagent which is used for precipitation? Has anyone worked with that reagent with urea containing samples?
Thanks
Joshua
Try to see this file for various concentration techniques
http://www.biolynx.ca/technical-support/on...n%20Methods.pdf
As history note: Many many time ago, when i was drived to extremity I 've applied the following: dialyse protein against PBS ( use Mw cutoff with pore limit more than 3 times smaller that Mw of your protein For ex for 24kDA in denatur form use 8kDa cutoff or may be till 3 kDa , because many proteins in denatur form are linear and their effective radius maller than native proteins). Then I bury dialyse tube with protein into solid high molecular PEG ( 20000 better 40000). PEG take water and concentrate sample. Make this procedure several times with fresh PEG. Be carefull during this procedure because very extensive drying can cause damage your dialyse tube . It was many many time ago......
Do you have possibility to make silver staining SDS PAGE ? It is really help you with diluted probes
http://www.biolynx.ca/technical-support/on...n%20Methods.pdf
As history note: Many many time ago, when i was drived to extremity I 've applied the following: dialyse protein against PBS ( use Mw cutoff with pore limit more than 3 times smaller that Mw of your protein For ex for 24kDA in denatur form use 8kDa cutoff or may be till 3 kDa , because many proteins in denatur form are linear and their effective radius maller than native proteins). Then I bury dialyse tube with protein into solid high molecular PEG ( 20000 better 40000). PEG take water and concentrate sample. Make this procedure several times with fresh PEG. Be carefull during this procedure because very extensive drying can cause damage your dialyse tube . It was many many time ago......
Do you have possibility to make silver staining SDS PAGE ? It is really help you with diluted probes
Hi, thanks for your advice but my problem is that I need to concentrate many (up to 8) samples each time I run a column for analytical purposes only. Your recommended procedure is too time consuming. I need a quick and easy concentration procedure and I don't care what happens to my protein as long as I can run it on SDS-Page.
Thanks
Thanks
your problem with precipitation may be because of the protein concentration. we used to do tca precipitations with the addition of tRNA (~1 ug, i think, may have been more) to help carry down the protein precipitate. it works well if you are coomassie staining or western blotting but you can't use this if you are silver staining (you get a big orange blob staining in your gel, so we abandoned this method when we will stain with silver). but, you may be able to find a different carrier that won't interfere with silver stain.
since you need to do a large number, do you have enough centricon filters? if so then you could use them and then clean the filter (as i said in my previous post).