Problem with Protein elution using GST sepharose 4B - (Sep/15/2009 )
Hello Everybody,
I am working on a protein which is supposed to be 10kD in size, I have tagged it with GST on Nterminal and His (6) on C terminal using pGEX4T1 vector.
This is the protocol I typically follow:
1) Inoculate about 2 ml of starter culture in 200ml LB + Amp and incubate at 37oC. Allow the OD 600 to reach to about 0.6-0.7. Remove 1 ml of culture for SDS PAGE analysis.
2) Induce with 0.2mM IPTG and incubate further at 25oC for 3-4 hrs.
3) Check the OD 600 once again after induction is complete. Remove one ml of culture for SDS PAGE analysis.
4) Pellet down 25 ml aliquots in 50 ml tube and store the pellet at -20oC
Protein Purification process:
1) Resuspend the cells in about 2ml of 1X PBS pH 7.3.
2) Add 0.1 volume of 10 mg per ml lyzozyme and 1X protease inhibitor cocktail (sigma)
3) Incubate at 37oC for about one hour.
4) Sonicate the cells at 32% amplititude for 10 secs at intervals of 30 sec for 40 secs.
5) Spin down at max. speed for 10 mins.
6) Collect the supernatant. Keep about 10 ul of supernatant for SDS PAGE analysis.
Column preparation and equilibration:
1) Take about 1.33 ml of slurry (GST sepharose) after mixing it properly.
2) Wash the slurry with about 5 ml of water twice. You can spin down at about 500-1000 rpm for 5 min.
3) Wash with about 5ml of binding buffer (1X PBS, pH 7.3) twice.
4) Add the supernatant to the equilibrated beads and allow for binding for at least 3-4 hrs on ice.
5) Spin down and collect supernatant (flow through)
6) Wash with about 5 ml of binding buffer and collect supernatant after spining down at 1000 rpm for 5 min (flow through).
7) Wash with about 5ml of binding buffer with 30 mM NaCl and collect supernatant spining down at 1000 rpm for 5 min (flow through).
8) Wash with about 5ml of binding buffer with 60 mM NaCl and collect supernatant spining down at 1000 rpm for 5 min (flow through).
9) Add about 5 ml of 30 mM reduced glutathione pH 8.0. Incubate it for about an hour on ice before collecting the flow through spining down at 3000 rpm for 10min .
11) Analyze the samples on 10% SDS PAGE gel.
Problems that I face:
1) The conc. of elute that I obtain is very less. The desired size of my protein is about 35 kD although I get a band at this size, I also get a prominent band at about 26 kD (wich is the size of plain GST)...
Dont if my protein is getting denatured..?
To confirm this I did a western blott with anti-His and got light up at both regions viz. 35 kD and 26kD region.
2) Most of the protein is bound to the column...? What can I do to increase the elution..?
3) Also, in case of one of the other proteins, I was sucessfull in eluting the fused protein of size 29kD. the target protein was about 4kD. But was not able to detect the protein after thrombin digestion.. I was able to see a band shift on gel. ..as in uncut migration was higher than the cut..but there was no correspoding band at 4 kD..? I tried doing western blotting and using anti His ...but it showed light up only in uncut...?
I am sorry if I am too elabarote about the procedure I have used..!!
Thank you very much i need to get this protein purified man..!!
regards,
Kaushik
Hello!
When u say that you obtain very little protein in the elutions...do u know for sure that your protein was adsorbed by the resine?, i mean, do u control the clarified extract before and after column with a +induction and -induction controls to be sure that you are controling the right band?, because it is possible that the binding of your protein was not efficient enough then your problem is different. For example, you can try adsortions of your protein with the resine for 1-2 h at RT which is more efficient or o/n at 4ºC which is less efficient but more sure regarding action of proteases.
"The desired size of my protein is about 35 kD although I get a band at this size, I also get a prominent band at about 26 kD (wich is the size of plain GST)..."
I have had the same problems of you with GST fusion. My opinion is that there is a protease target next to GST which leaves the His-tag fused to the GST. I have produce several recombinant proteins fused with GST and in the 75% of the cases i obtain this annoying band.
"the target protein was about 4kD. But was not able to detect the protein after thrombin digestion.. I was able to see a band shift on gel. ..as in uncut migration was higher than the cut..but there was no correspoding band at 4 kD..? I tried doing western blotting and using anti His ...but it showed light up only in uncut...?"
May be this is a stupid question but....this protein (4 kDa) is really small, there is any possibility that you are loosing your band in the SDS-PAGE, even in a 17% acrylamide gel. If i remember ok the smaller Mw in my case is 7 kDa.
Good Luck
KAUSHIK THAKKAR on Sep 15 2009, 04:21 AM said:
I am working on a protein which is supposed to be 10kD in size, I have tagged it with GST on Nterminal and His (6) on C terminal using pGEX4T1 vector.
This is the protocol I typically follow:
1) Inoculate about 2 ml of starter culture in 200ml LB + Amp and incubate at 37oC. Allow the OD 600 to reach to about 0.6-0.7. Remove 1 ml of culture for SDS PAGE analysis.
2) Induce with 0.2mM IPTG and incubate further at 25oC for 3-4 hrs.
3) Check the OD 600 once again after induction is complete. Remove one ml of culture for SDS PAGE analysis.
4) Pellet down 25 ml aliquots in 50 ml tube and store the pellet at -20oC
Protein Purification process:
1) Resuspend the cells in about 2ml of 1X PBS pH 7.3.
2) Add 0.1 volume of 10 mg per ml lyzozyme and 1X protease inhibitor cocktail (sigma)
3) Incubate at 37oC for about one hour.
4) Sonicate the cells at 32% amplititude for 10 secs at intervals of 30 sec for 40 secs.
5) Spin down at max. speed for 10 mins.
6) Collect the supernatant. Keep about 10 ul of supernatant for SDS PAGE analysis.
Column preparation and equilibration:
1) Take about 1.33 ml of slurry (GST sepharose) after mixing it properly.
2) Wash the slurry with about 5 ml of water twice. You can spin down at about 500-1000 rpm for 5 min.
3) Wash with about 5ml of binding buffer (1X PBS, pH 7.3) twice.
4) Add the supernatant to the equilibrated beads and allow for binding for at least 3-4 hrs on ice.
5) Spin down and collect supernatant (flow through)
6) Wash with about 5 ml of binding buffer and collect supernatant after spining down at 1000 rpm for 5 min (flow through).
7) Wash with about 5ml of binding buffer with 30 mM NaCl and collect supernatant spining down at 1000 rpm for 5 min (flow through).
8) Wash with about 5ml of binding buffer with 60 mM NaCl and collect supernatant spining down at 1000 rpm for 5 min (flow through).
9) Add about 5 ml of 30 mM reduced glutathione pH 8.0. Incubate it for about an hour on ice before collecting the flow through spining down at 3000 rpm for 10min .
11) Analyze the samples on 10% SDS PAGE gel.
Problems that I face:
1) The conc. of elute that I obtain is very less. The desired size of my protein is about 35 kD although I get a band at this size, I also get a prominent band at about 26 kD (wich is the size of plain GST)...
Dont if my protein is getting denatured..?
To confirm this I did a western blott with anti-His and got light up at both regions viz. 35 kD and 26kD region.
2) Most of the protein is bound to the column...? What can I do to increase the elution..?
3) Also, in case of one of the other proteins, I was sucessfull in eluting the fused protein of size 29kD. the target protein was about 4kD. But was not able to detect the protein after thrombin digestion.. I was able to see a band shift on gel. ..as in uncut migration was higher than the cut..but there was no correspoding band at 4 kD..? I tried doing western blotting and using anti His ...but it showed light up only in uncut...?
I am sorry if I am too elabarote about the procedure I have used..!!
Thank you very much i need to get this protein purified man..!!
regards,
Kaushik