Hi everyone .....
I am trying to purify and isolate a protein of about 1.3Kd size....(well almost a peptide at that ).....i need to get enough pure to be able to sequence it ......Any ideas how to go about doin this ???.....Are there any gels or something I can run to catch it on ???....Am trying a 20% SDS Gel but so far no luck .....

You may want to try the Slide-A-Lyzer 2K MWCO Dialysis Cassettes from Pierce. Or you can take a look at this Pierce Selection Guide

Otherwise here are some pretty good overviews of some other protein purification methods:
Protein Purification

Protein Purification - Principals & Practices

Protein Purification Handbook from amersham pharmacia biotech


If you have funding available this text may be of some help -
Guide to Protein Purification

What techniques have you tried so far?

hey everyone thanks for your help so far . the links are helpful .
so far i have put it through a Seph Q folowed by a CM Seph Column and followed by a Phenyl Seph Column ......but havent gotten it completely pure yet .

Hi trook ...
so i am new to this and slightly confused , hopefully you can bear with my questions...
wouldnt the cassette you suggested retain everything bigger than 2000 (and 63% of a 1350 size protein ..which is similar to the size of mine ) ......how would this help me purify then ?

or did you mean that i should try a reverse dialysis and put high conc outside cassette and try and catch my protein inside it ???...

thanks .

hey ...thanks .....yes that is wht i am trying to do now .....isolate it on a C-18 column....keeping my fingers crossed ......

This might sound too simple - but I've purified small (not as small as 1kd) DNA & proteins this way before. Run your protein sample on the appropriate concentration PA gel. Cut out the section of the gel showing the protein. Place gel piece in a dialysis bag with tank buffer. Place dialysis bag in electrophoresis tank with tank buffer and run current until the protein runs off the gel. You now have your protein in the dialysis bag with tank buffer. To concentrate the protein - place the bag in PEG for a while. This is one way of doing it.
Its simple and I hope it helps.

This might sound too simple - but I've purified small (not as small as 1kd) DNA & proteins this way before. Run your protein sample on the appropriate concentration PA gel. Cut out the section of the gel showing the protein. Place gel piece in a dialysis bag with tank buffer. Place dialysis bag in electrophoresis tank with tank buffer and run current until the protein runs off the gel. You now have your protein in the dialysis bag with tank buffer. Remove the piece of gel.To concentrate the protein - place the bag in PEG for a while. This is one way of doing it.
Its simple and I hope it helps.

thanks frog egg .. will try this out ...
by the way... i think i managed to catch it on a 18% SDS gel ....using silver stain....still have to chk with a couple of assays though .....

Do you have sequence information for your protein? If it's that small, you could have the peptide synthesized, then couple it to KLH and make antibodies. Life is always simpler once you have an antibody to work with!

Wouldn't it be possible to just centrifuge it through one of the smallest MWCO Millipore filters? I used to do this for my 6Kd protein since the other proteins were all larger.

Hi all...
thanks for your suggestions !..
i did use millipore filters ..used a 3,000 NMWL filter and that helps ....now am going to try and transfer it to a PVDF memebrane and try and sequence it ......
also i was you have mentioned KLH ?...i was wondering what that is ?..
thanks

KLH stands for Keyhole Limpet Hemocyanin. It's a large, immunogenic protein that carries oxygen in molluscs. It's used as a carrier protein to couple peptides/small proteins to so that they will elicit a strong immune response and generate antibodies when injected into a rabbit, goat, mouse, etc.