My GF buffer which i solubilize my protein
0.05M Tris CL
0.05M Nacl
0.02% sodium azide
pH 8.0
is there a problem with this buffer??
is the Nacl too low??
There's suggestion of using higher Ionic strength:
0.010 M tris
0.8M nacl
0.1mM EDTA
7 mM p-ME
0.01% triton-x
pH 7.5
or should i change to this??
pI of my protein is 6.99 theoretically.
I run my column with my GF buffer at 1ml/min
bed height 27cm..cross area 0.79
bed volume would be ~23 ml.
Loaded 1 ml of 12 mg/ml BSA (just to see the packing)
result :
I don't see a peak at all.
what is wrong???
then : i clean the column with 0.5M Naoh
and GF buffer
BUT>>>> after a volume of GF buffer pass through... the buffer can't seem to pass through .. i tried and waited.
is it because of the 0.5M naoh??
should i wash with NACL instead??
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