How do you go around concentrating and desalting with minimal sample loss? - (Sep/13/2006 )
Hello,
I wanted to initiate this discussion as it is one of the most challenging aspects while working with proteins!
As you are aware that current protein concentration, dialysis and desalting methods are time consuming and laborious and may result in valuable loss of a significant portion of the protein sample jeopardizing the detection of low abundant proteins.
What is your approach to solve such issue?
My favorite is amicon. it's filtration through a size exclusion membrane, by centrifugation. It goes fast, allows concentration and buffer exchange if repeated several times. however you lose proteins, especially if you have few proteins. that's why i reuse these columns.
For desalting, (buffer exchange) I prefer PD-10 columns, it's a gel filtration. I think I loose less proteins, but there is a dilution factor of 1.5 at least. it depends on the volume of the sample and the volume of the column I use.
i agree with missele. the easist way to concentrate protein is in a concentration column. but i know someone who lyophilises his protein regularly. of course his protein is stable and retains activity after freeze-thaw cycles.
As you have mentioned Missele one potential problem with column based desalting is resulting in sample dilution and thus may reduce the potential for detection of low abundant proteins. The other limitations with filtration columns is sample loss (how much sample loss do you get?), cut off point, and potentail binding for highly hydrophobic proteins to the filtration membrane.
The problem with Lypholyzation is that it does not work well with lprotein samples with low volumes and as Soraya mentioned works well for highly stable proteins but not for all proteins.
I was wondering if members of the forum has used similar methods and what worked and what did not work for them!!!
The problem with Lypholyzation is that it does not work well with lprotein samples with low volumes and as Soraya mentioned works well for highly stable proteins but not for all proteins.
I was wondering if members of the forum has used similar methods and what worked and what did not work for them!!!
the loss depends on the protein and the concentration : from 5 to 50%.
one solution could be to block the membrane with BSA before to concentrate your protien.
Hi Missle,
What percentage of BSA you are using and would it that add to the complexity of the protein or proteins being concentrated?
Actually there is aproduct currently offred by Norgen Biotek under the name of ProteoSpin CBED, basiclly it stands for simultanuous Concentration, Buffer Exchange and Desalting of the protein samples on the same column with minimal loss of sample (http://www.norgenbiotek.com/index.php?id=pkitpro)
I was wondering if you had your self or any member of the forum an experience with such technology for protein sample clean-up and detergent removal especially for mass spec analysis and like to share in this thread?
thanks!
I think that a 1% solution is enough. you concentrate it, and then you wash the upper part (where the BSA will stays, and where you will recover your protein later), but you have to keep in mind that you could contaminate your sample with BSA. It depends what you want to do. You can also use an other protein. (Personnally I never did such improvements, and never controled if BSA will contaminate my sample)