Problem with GST tagged baculovirus protein - (Oct/23/2008 )
Hi -
I have a soluble GST fusion protein expressed in the pGEX-4T-1 vector in baculovirus cells. I am able to get the protein soluble from the cells but I cannot bind it to the glutathione resin for some reason. I tried binding it at 4 degrees rocking using lysis buffer (500 mM NaCl, 20 mM Tris, 1 mM EDTA, 10 mM BME, 0.1% sodium deoxycholate, 15 mM KCl, and protease inhibitors, pH 6.8) but all the protein came out in the flow through. I tried binding for about 2 hours. Next I reduced the salt concentration by diluting down with a phosphate buffer (50 mM sodium phosphate, 1 mM EDTA, 0.01% triton, 1 mM BME, pH 6.8) down to 150 mM NaCl but it still came out in the flow through. Now I took the flow through that didn't bind and I'm trying to bind it at 4 degrees overnight but I am not too hopeful at this point. The glutathione sepharose is from GE and the troubleshooting for the product is not any help (I'm not sonicating the cells, I have BME in the buffer, I equilibrated the resin before use, and its fresh resin so that is all they suggest). Can anyone help???
Hi,
I have the same problem with GE glutation. I bougth the kit and I am not sure what is wrong. I am writing to their technical support maybe they can helt. I am thinking maybe there is some thing wrong with their product. If you plan to contact them tell me their reply! It is a good idea if we keep in touch!
Cheers,
Sara
I have a soluble GST fusion protein expressed in the pGEX-4T-1 vector in baculovirus cells. I am able to get the protein soluble from the cells but I cannot bind it to the glutathione resin for some reason. I tried binding it at 4 degrees rocking using lysis buffer (500 mM NaCl, 20 mM Tris, 1 mM EDTA, 10 mM BME, 0.1% sodium deoxycholate, 15 mM KCl, and protease inhibitors, pH 6.8) but all the protein came out in the flow through. I tried binding for about 2 hours. Next I reduced the salt concentration by diluting down with a phosphate buffer (50 mM sodium phosphate, 1 mM EDTA, 0.01% triton, 1 mM BME, pH 6.8) down to 150 mM NaCl but it still came out in the flow through. Now I took the flow through that didn't bind and I'm trying to bind it at 4 degrees overnight but I am not too hopeful at this point. The glutathione sepharose is from GE and the troubleshooting for the product is not any help (I'm not sonicating the cells, I have BME in the buffer, I equilibrated the resin before use, and its fresh resin so that is all they suggest). Can anyone help???
I have the same problem with GE glutation. I bougth the kit and I am not sure what is wrong. I am writing to their technical support maybe they can helt. I am thinking maybe there is some thing wrong with their product. If you plan to contact them tell me their reply! It is a good idea if we keep in touch!
Cheers,
Sara
I have a soluble GST fusion protein expressed in the pGEX-4T-1 vector in baculovirus cells. I am able to get the protein soluble from the cells but I cannot bind it to the glutathione resin for some reason. I tried binding it at 4 degrees rocking using lysis buffer (500 mM NaCl, 20 mM Tris, 1 mM EDTA, 10 mM BME, 0.1% sodium deoxycholate, 15 mM KCl, and protease inhibitors, pH 6.8) but all the protein came out in the flow through. I tried binding for about 2 hours. Next I reduced the salt concentration by diluting down with a phosphate buffer (50 mM sodium phosphate, 1 mM EDTA, 0.01% triton, 1 mM BME, pH 6.8) down to 150 mM NaCl but it still came out in the flow through. Now I took the flow through that didn't bind and I'm trying to bind it at 4 degrees overnight but I am not too hopeful at this point. The glutathione sepharose is from GE and the troubleshooting for the product is not any help (I'm not sonicating the cells, I have BME in the buffer, I equilibrated the resin before use, and its fresh resin so that is all they suggest). Can anyone help???
I had the same problem before, but the solution is simple. The suggested bed volume used for a given culture volume in the GE protocol is not that optimal at least for my protein. I used 10 times more bed volume than suggested and it works!