I tried to purify using anion exchange column (High Q), i got a peak (intensity >1500mAu). But when i did 15% SDS gel...i didn't get any band from those collected fractions. But my crude extract showed band. Also while performing dialysis i could see that my protein is precipitating.
Hello! Tell me about your buffers? Are you using HEPES, Tris, or PBS? What pH? How much NaCl and what was the gradient range off the exchange column? Do you have 10% glycerol? Do you have a reducing agent in your buffer? What are the MWCO for your rough dialysis and what equipment are you using, tangential flow? Finally, do you have access to a dynamic light scattering instrument? You may not need one unless you are trying to produce mass quantities. If you put the amino acids into ExPASy ProParam it will give you a calculated pi which you can use to decide anion or cation, but buffer issued come first. You may even need some tween or DDM detergent.
Hello sir! Thank you for your reply.
tris-HCl pH 8.0
50mM - 2M NaCl gradient range
i used refolding buffers (3 buffers having diff urea conc 8M, 4M, 2M)..it consists of 10%glycerol, reduced and oxidized glutathione
yes i have added 100mM DTT as reducing agent
6-8kDa MWCO...was used for dialysis
i have access to DLS but sir i am not interested in producing mass quantities.
Hello! I must say that I don't know much about preparation of refolded proteins as I have always purified proteins from non-denaturing conditions. So I forgot to ask how you were expressing the protein, insect cells, E. coli, yeast, or getting it from tissue. This will effect the purification procedure. Tris is a perfectly good buffer and pH of 8.0 doesn't seem to unreasonable. It may be better to process the the raw extract with pH 7.8 Tris 10mM with 150 mM of NaCl, 10% glycerol, 1mM DTT (or 6mM bME) then buffer exchange into your pH 8.0 Tris with lower salt for the High Q gradient. After try to buffer exchange back into 10 mM Tris pH 7.5 with 150mM NaCl 10% glycerol and 1mM DTT. Also we usually add some PMSF and E64 to the cell lysate to inhibit proteases. If E64 is too expensive just PMSF (serine protease inhibitor) is fine. Also which species BMP2 are you purifying? If you can put the amino acid sequence into this web calculator and it will calculate a pI for you perhaps cation exchange will work better. http://web.expasy.org/protparam/
Sir i am expressing the protein in E. coli. Yes sir i added PMSF and lysozyme. Its human BMP-2 sir. Isoelectric point is 7.96.
I think that your buffer may be too close to the isoelectric point of the protein. If the pI really is 7.96 and you are using pH 8.0 buffer you should have no charge on the protein to bind the column matrix. Please look at this handbook and especially page 36. This is pretty much the best info for ion exchange chromatography. I quick read will get you where you need to be. Also the DTT is probably too high if 100mM. Here is the link, http://wolfson.huji.ac.il/purification/PDF/IonExchange/AMERSHAM_iIEXandChromatofocManual.pdf Good Luck!!
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