Hello,
PMSF inhibits only 1 class of proteinases
You can try to add other inhibitors :
e.g. for purification from yeast I used the following in addition to PMSF:
1000 x stock individual inhibitors
pepstatin5 mg/ml methanol
chymostatin1 mg/mlDMSO
antipain3 mg/mlmethanol (sonicate to dissolve)
aprotinin2 mg/mlwater
leupeptin1 mg/ml water
E-642 mg/ml50 % ethanol
bestatin5 mg/mlmethanol
as well as EDTA for metallo-proteases
Some suppliers like Roche sell pre-made mixtures like protease inhibitor
pills.
Also be as fast as possible with all steps and keep on ice
Best of luck,
Ruud
On 26/10/17 16:18, Anamika Singh wrote:
Dear All,
I am purifying the His-tagged proteins ( 21Kda) using buffer 20mM
Tris, 150mM Nacl, and 5mM MgCl2 with 500mM imidazole. Earlier I was
facing the precipitation problem during dialysis but with the addition
of 50mM L-Arg somehow managed to overcome the precipitation issue. But
this time I have seen after overnight dialysis there are degradation
products on SDS page. I have used protease inhibitor (PMSF) in my
sonication buffer.
Please suggest me to overcome this problem.
Thanks
--
Anamika
--
Ruud Hovius
EPFL SB ISIC LIP
BCH 4209
CH-1015 Lausanne
+41-21-693-9442
http://lip.epfl.ch
