最近用GE healthcare的glutathione sepharose 4B resin purify a couple of GST- tagged transcription factor DNA binding domains. The protein were bound to the beads with high efficiency. However, when I used glutathione buffer to elute the GST-protein off the beads, they can't be eluted. I tried 10 mM ,50 mM and 100 mM glutathione. To make the buffer, I diluted 1 M Tris (pH 7.5 at room temperature) to 50 mM, and added glutathione powder. There was also 2 mM DTT in the buffer. The elution was carried out at 4 degree where Tris should be at pH 8.0. I tried different resin and different glutathione, but none of them worked. So the problem must be in the buffer conditions. Does anyone know what is the potential problem? pH? I never measured pH of the glutathione-Tris buffer.
【在 f*****s 的大作中提到】 : 最近用GE healthcare的glutathione sepharose 4B resin purify a couple of GST- : tagged transcription factor DNA binding domains. The protein were bound to : the beads with high efficiency. However, when I used glutathione buffer to : elute the GST-protein off the beads, they can't be eluted. I tried 10 mM ,50 : mM and 100 mM glutathione. To make the buffer, I diluted 1 M Tris (pH 7.5 : at room temperature) to 50 mM, and added glutathione powder. There was also : 2 mM DTT in the buffer. The elution was carried out at 4 degree where Tris : should be at pH 8.0. : I tried different resin and different glutathione, but none of them worked. : So the problem must be in the buffer conditions.
50 also . the buffer pH to 8.0 after dissolving it in Tris.t be in the buffer conditions.
【在 f*****s 的大作中提到】 : 最近用GE healthcare的glutathione sepharose 4B resin purify a couple of GST- : tagged transcription factor DNA binding domains. The protein were bound to : the beads with high efficiency. However, when I used glutathione buffer to : elute the GST-protein off the beads, they can't be eluted. I tried 10 mM ,50 : mM and 100 mM glutathione. To make the buffer, I diluted 1 M Tris (pH 7.5 : at room temperature) to 50 mM, and added glutathione powder. There was also : 2 mM DTT in the buffer. The elution was carried out at 4 degree where Tris : should be at pH 8.0. : I tried different resin and different glutathione, but none of them worked. : So the problem must be in the buffer conditions.
【在 f*****s 的大作中提到】 : 最近用GE healthcare的glutathione sepharose 4B resin purify a couple of GST- : tagged transcription factor DNA binding domains. The protein were bound to : the beads with high efficiency. However, when I used glutathione buffer to : elute the GST-protein off the beads, they can't be eluted. I tried 10 mM ,50 : mM and 100 mM glutathione. To make the buffer, I diluted 1 M Tris (pH 7.5 : at room temperature) to 50 mM, and added glutathione powder. There was also : 2 mM DTT in the buffer. The elution was carried out at 4 degree where Tris : should be at pH 8.0. : I tried different resin and different glutathione, but none of them worked. : So the problem must be in the buffer conditions.
g*1
10 楼
Check the pH. Glutathione can drop pH to quite low. And bound protein can not be eluted.
f*s
11 楼
I've run SDS-PAGE so many times. The protein is clearly still on the beads. I'll check the pH today. I just didn't think 10 mM glutathione can change the pH of 50 mM Tris significantly.
s*e
12 楼
is there protease cutting site between ur proten and GST? if yes u can try cut it from GST w/o elution.