I have tried with 20% PEG, 100mM acetate buffer and ammonium sulphate (100mM to 400mM), incubate at 17 degree overnight but crystal did not form.
@Ravi Ananth i want to study the interaction of drug with lysozyme. I my reaction mixture Gucl is present, which is generally added to unfold the protein. I will form the crystal in the presence of drug+Gucl+Lysozyme and then diffract it to reveal the amino acid specific interaction between drug and protein.
"then diffract it". Do you mean X-ray diffraction? If so what equipment do you use at IIT K? I've done some work with the folks at the Bose Institute Kolkata on Lysozyme crystals. Just curious.
Once you have your diffraction patterns, we can compare maybe? What detectors do you have access to in your XRD lab?
Hi Mangesh,
I do not know your experimental setup, but 4 M GuHCl is already pretty strong. Are you sure that the protein is still folded under these conditions? At low pH lysozyme is already unfolded at 4 M GuHCl and at neutral pH it is very close to the transition. See these papers: http://onlinelibrary.wiley.com/doi/10.1110/ps.041085005/full
and
http://www.jbc.org/content/249/17/5388.short
Also, 4 M GuHCl increases the proteins solubility dramatically. So I doubt that you will get a supersaturated solution with the lysozyme.
When you want to study the interaction of a drug with lysozyme - what do you want with the GuHCl?
Best regards
Assuming your protein is not denatured (in which case you can't get crystals), overnight is not always long enough to get crystals. In fact, it rarely is, particularly if your solution increases the protein solubility as someone above mentioned. You can try increasing the protein concentration or precipitant concentration if this is the case. When you view your drop under a microscope is it clear or do you see light precipitate?
Also why must you have GuHCl? It seems very unlikely that the protein won't be denatured under those conditions
Are you sure about 4 M guanidinium-HCl? You might be able to do it with 4 M urea and people have a tendency to mix up those two.
Why are you trying to crystallize under those conditions though? And if it is Hen Egg White Lysozyme then why do you have a drug for interacting with egg white? Or is this one of the human lysozymes?
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