Gel filtration - problem! (Aug/14/2008 )
HI there. I am a masters student working on purifying a novel membrane receptor. I have successfully solubilized the receptor and shown that it is functional in a bio assay. I hoped to purify the protien using gel filtratoin using a 300 mwco gel filtration colum. Problem is that after running the sample through the column i lose the activity! I dont know why gel filtratoin would render my receptor unfunctoinal. Any suggestions as to why this may be happening. My buffer (running and sample) is HBSS containing 20mg/ml CHAPS. Any help is greatly appriciated! Thanks!
Try running the gel with your assay buffer? Are there components of your assay buffer needed for receptor binding that are not in your gel running buffer? Also, are you running the column at 4C?
My running buffer is exactly the same as my assay buffer (minus the solubillzed protein obviously). I am not running the column at 4 deg, i have been using an HPLC at room temp, however i have just today set up a column in a cold room and will be trying it (not hplc though).
ok i tried doing it in the cold room at 4 deg and still lost all my activity. I also tried concentrating the samples and still got nothing. Does anyone have a guideline i could use as to how much protein of interest should be added to a column to overcome potential loss effects?
you need to test to see if your protein stick to the column though nonspecifci adhesion. 300 mwco wont do much separation other than desalting.
Ok im not sure how i would test if my protein is binding non specifically to my column, can you walk me through a test for that? Also why is 300 not a good seperator? should i use a higher or lower column size?
with regard to non-specific binding, it normally occurs when you have low protein concentration to start with. Proteins with hydrophobicity, like the ones you are working with may be more likely to stick to the column. You need to follow the protein distribution in the fractions by OD280, dye-based protein assy, SDS-PAGE, or activity etc. Add some salt (not too high) in the buffer will reduce such non-specific interaction.
Be sure that you read some background info on gel filtration technique, then you would understand why MWCO300 is no good. It has nothing to do with the size of the column.
Be sure that you read some background info on gel filtration technique, then you would understand why MWCO300 is no good. It has nothing to do with the size of the column.
I am aware of the protein concentration as i do OD280 (well the machine does) and dye based and SDS. I get one main peak and a few other little bumps. Maybe the s300 is why im only really getting one main peak, but im having troubles finding suggestions on a proper size. I will add salt to my buffer, currently i use a 300mM salt concentration. Thanks for your help
I am also now realizing that my gel filtration may not be as efficient as i may have thought. I get one large peak at about fraction 7 (of 15) that has a few bumps at the top, drops steeply and then tails off. (should i post a pic?). Any suggestions of what the problem may be?
hi there...i wonder somehow if you are using calcium and magnesium free hanks solution....and also if the pH of this is 7.2.....also the are you adding HEPES in HBSS and also using CHAPS? or any one of them?....i think you migt find the answer to your problem somewhere here...