I agree to this last suggestion. For one of my protein, I had to add at
least 10 % of glycerol in my buffers to keep my protein stable while
purifying. For crystallization, I diluted it to 20 % glycerol (vapour
diffusion method) which allowed it to be stable in the crystallization
drop and eventually leading to crystallization. (well, again it depends
from protein to protein).
Another thing you could try for bigger crystals is to increase your
protein concentration, (if it is stable enough) and set up at 4 C (rather
than 16 C).
Hope this helps,
Manish
> Sorry - this may have been mentioned previously, but have you tried
> banging in some glycerol (5-10%)?
>
> J
>
> shivesh kumar <[log in to unmask]> wrote:>
>> Dear all
>> I welcome all the suggestions regarding my crystals which is coming with
>> the
>> precipitation.The pI of the protein is 4.2 and the drop precipitates
>> with
>> MPD as low as 30% within 4-5 hrs.I am trying the pH ranging from 3.6-5.2
>> at
>> 16C.There is no precipitation at 20% of MPD.Also,at high conc,the
>> protein
>> precipitates.The crystals are coming as florets as I mentioned with
>> 2+2.Should I go for higher pH like uptp 8.5 or so.crystals are not
>> growing
>> bigger in size so that I can mount.Should I try changing the
>> temperature.Ican use 4C and
>> 16C.Right now I am keeping plates at 16C.I am concentrating the protein
>> through lyophilization.As soon I pool it for Gel filtration,majority of
>> it
>> get precipitated,not soluble protein.Is there any method to make the
>> protein
>> stable?
>> I appreciate all the informative suggestions.Thanx in advance and thanx
>> everybody who has suggested the various options.
>> Shivesh kumar
> --
> Professor James Whisstock
> NHMRC Principal Research Fellow / Monash University Senior Logan fellow
>
> Department of Biochemistry and Molecular Biology
> Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia
> +613 9905 3747 (Phone)
> +613 9905 4699 (Fax)
> +61 418 170 585 (Mobile)
>
*************************************************
Manish B. Shah, PhD.
Postdoctoral Fellow
Hauptman-Woodward Medical Research Institute
700 Ellicott Street
Buffalo, NY 14203.
*************************************************
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