Dear Gauri
This may be the saturation point of your protein and beyond this it will start
precipitating in a particular buffer. Try to set the crystallization trial (if
it is your application ) and select those condition in which you see clear
drop.
Change the purification buffer with that
use the following procedure.
1) immediately after use pass freshly prepared cold reduce glutation 20mM ,
1mM
EDTA in 50mM tris ph8.0. 2-3 CV,
2) wash alternate with buffer 1 and buffer 2 (GE lifescience), 2 cv, 4-5 times,
then 1M Nacl 2-3 cv
3) wash with pbs , 5 CV
4) wash with 1% triton,
Dear all
Thanks for yours valuable suggestion.
Just some addition information to make the query clear.
1)My protein has 14 cysteine residues.
2)It is not a metal binding protein.
3)I have added the protease inhibitor cocktail + PMSF during sonication and
cleavage. I saw only one band of
I am working on 45 kd protein (pI 5.3) for crystallization . It is in pGEX
vector.I do the expression (induction 24 degree 16 hrs) and purification from
Rosetta 2 DE3 cells (has additional rare codon).when I run the affinity
chromatography purified protein (cleaved protein after removal of
sometime it does happen becoz of protein aggregation, or reducing environment.
but it may be your protein color as well that visible during concentration
Vikrant
From: sandeep toskgu...@rediffmail.com
To: CCP4BB@JISCMAIL.AC.UK
Sent: Fri, 24
I want to do cloning of a 40 Kd protein in pRSETA, and pGEX-KT vector. I don't
have any idea about protein solubility, its multimeric form, stability and
disorder etc. There is nothing known in the literature also. Is there any
software that can predict these parameters, so that i can decide
Dear all
I am working on purification of 14 kd protein(pI 8.3, basic protein) that has
MBP(maltose binding protein, 45 kd,) tag, and same protein in other
vector(pGEX-KT) that has GST tag. During affinity purification in both cases I
used 300mM nacl and 50 mM tris, pH 7.5 buffer throughout
