Hi all
I am trying to isolate a seed protein. I did ammonium sulphate precipitation followed by dialysis against MilliQ to remove the salts. After dialysis, the protein is getting too precipitated. Even after more than 8 times dilution, I am unable to get a clear solution. I have to load this on Sephadex G 100 for SEC.How do I proceed??
Thanks
I don't have any personal experience purifying proteins from seeds, but I wonder whether the precipitated material you are seeing may be starch. If so, or if it is protein other than the protein you are trying to purify, remove the precipitate by centrifugation before the chromatography step.
If the protein you want is in the precipitate, you may be able to reduce the amount of precipitation by buffering at a different pH and/or maintaining the protein at a different salt concentration. Alternatively, collect the precipitate by centrifugation and try to get it to dissolve in low or high salt, low or high pH.
Have you ever tried not to dialyze against pure water but rather against the buffer you are going to use for SEC? Solutions can change their pH upon dilution.
don't do with dialysis against milliQ. if you just want to remove salt, then do dialysis against with buffer with no salt and again don't do dialysis at once. for example, in your precipitate protein, salt conc. is 500mM. Then, first dialysis u should do with your protein buffer plus 350 mM salt for at least 3 hours. then second dialysis, buffer with 200mM salt,then 125mM, 75mM, 25mM and then 0mM. you have to follow sequential step in dialysis. If you will jump directly from height, more chances for accident. same things with protein, so do sequentially and u will see...yr protein will be smiling and give expected results.
Good luck....
Hi Shreyasi, I agree with the above comments about not dialyzing your protein against MilliQ.
If you do not want salts in your protein prep., you can dialyze against volatile buffers, like ammonium acetate or ammonium bicarbonate. You can then lyophilize your protein.
Since the next step you are planning after dialysis is SEC, you can keep as much salt as necessary in the dialysis buffer to keep the protein in solution. After SEC, the buffer will be whatever the column was equilibrated and eluted with, as long as the proteinis large enough that it elutes ahead of the salt. The same goes for pH and buffer type.
Certain fractions of the seed storage proteins are soluble in salt solution i.e. thye globulin fractions of the SSP are considered to be soluble in salt solutions, maybe they are precipitating out.
Seeds tend to have high amounts of storage proteins. A common way to isolate these proteins is to adjust the pH to the pI and see large amounts of protein will come out of solution. You might be working close to the pI for those storage proteins. You will want to work at a different pH. Another thing you might try if your desired protein is soluble in water (no salts) is to make your protein extract in water. Many seed storage proteins will not be extracted.
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