I would like to do EMSA with a protein that has an isoelectric point of 9.3. I've been trying native PAGE changing buffers pH and also a variant with Ponceau and none of them seems to work fine for me. Does anybody have any protocol that worked for you? Or any suggestion? Thank you very much for your time!
In native PAGE, your protein has to have the necessary charge to move into the gel - if the pI of your protein is higher than the pH in your gel, or if the net charge is 0 (zero), your protein will move in the wrong direction, or not at all.
As suggestion, you might try running a typical native PAGE but when you put the electrodes into the power supply, invert them (i.e., put the red electrode into the socket for the black power supply and black electrode into the socket for the red power supply). Basic proteins will not migrate into a standard PAGE gel because only proteins with lower pIs (say < 7-8) will migrate under native conditions. Acidic proteins are negatively charged and will migrate into a standard native page, but basic proteins are positively charged and will not migrate into a standard native page...basic proteins migrate OUT of a standard native page. If you flip the electrodes, you reverse the electric field, and this will allow basic (i.e., positive) proteins to run into the gels. BioRad recommends this approach for native-PAGE of basic proteins (see their manual for TGX mini-gels under "native-PAGE". This should work with any standard native gel type, whether it's BioRad's gels, some else's gels, or hand-case gels.
In native PAGE, your protein has to have the necessary charge to move into the gel - if the pI of your protein is higher than the pH in your gel, or if the net charge is 0 (zero), your protein will move in the wrong direction, or not at all.
As suggestion, you might try running a typical native PAGE but when you put the electrodes into the power supply, invert them (i.e., put the red electrode into the socket for the black power supply and black electrode into the socket for the red power supply). Basic proteins will not migrate into a standard PAGE gel because only proteins with lower pIs (say < 7-8) will migrate under native conditions. Acidic proteins are negatively charged and will migrate into a standard native page, but basic proteins are positively charged and will not migrate into a standard native page...basic proteins migrate OUT of a standard native page. If you flip the electrodes, you reverse the electric field, and this will allow basic (i.e., positive) proteins to run into the gels. BioRad recommends this approach for native-PAGE of basic proteins (see their manual for TGX mini-gels under "native-PAGE". This should work with any standard native gel type, whether it's BioRad's gels, some else's gels, or hand-case gels.
Anyway, here is a Bio-Rad protocol for analysis of basic proteins in native gels
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2376.pdf
If your protein has such a high isoelectric point maybe you can try one of this options:
1) you can try with Blue native electrophoresis, on this system you use Coomassie for loading the protein into the gel, the Coomassie gets attached to the protein and
this may help to the protein to get into the gel. (A Practical Guide to Membrane Protein Purification. Volume 2 in Separation, Detection, and Characterization of Biological Macromolecules. 1994, Pages 59–79)
2) You can keep the same system for the electrophoresis and just exchange the electrodes for the protein which normally won't get into the gel will separate.
3) you can run the electrophoresis with different buffers and look for the better resolution on your proteins: This paper will help you if you try this option (Anal Biochem. 1982 Oct;126(1):94-9.
Electrophoresis buffers for polyacrylamide gels at various pH. McLellan T.)
Hi Sara. Were you able to get this to work? I'm also currently working with a protein of a similar pI
Hi Shruti. Finally I managed to have a decent gel with native blue electrophoresis. Good luck!
Hi Sara, I would like to run a native gel for my membrane receptor protein which has a pi of 8.5. I have been looking at many recipes and stumbled on your post. could you please share your protocol and tips with me?
Hi Gurjot,
I've found a paper that can be usefull for you: Schägger H, von Jagow G 1991 Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–231.
I was following the paper called "Blue Native Polyacrylamide Gel Electrophoresis: A Powerful Tool in Diagnosis of Oxidative Phosphorylation Defects"
Good Luck!
Hi Sara,
I am currently working with Native structures of Egg and pea proteins as a part of my Masters project. Can you please share your protocol for Native PAGE?
Hello, I would like to know which is the best protocol to extract the protein before conducting the PAGE protocol. I was thinking grind the tissue with Liquid Nitrogen and the add the grinding buffer, or do you think its better to grind the plant tissue directly with the buffer instead of using the nitrogen?
Thanks!
I suppose the author of this question might not be working on this anymore/found a solution, but for anyone facing the same problem(s) I suggest reading up on Invitrogen's/Novex BN-PAGE protocols. I was lucky enough to have the Invitrogen equipment in my lab, but BN-PAGE could probably be run on the BioRad minigel system too.
The point of Blue Native is to use Coomassie to impart negative charge on your proteins without denaturing them (like SDS would in SDS-PAGE). I recently got alright results with pI ~9 protein.
I would personally really recommend using their sample preparation kits. In my case, I used this together with a native plasma membrane extraction kit.
Gaber, I would recommend you post a new question about purification and WB - that's not really the topic here?
can any one kindly let me know how the problem was solved How commassie was adapted to EMSA for movement of protein from well into the gel . any help will be highly appreciated .
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