6 replies to this topic

[biolcrazy]

I am using the Pierce GST spin purification column for purifying my GST tagged protein.  I have tried using the B-PER reagent/ sonication for bacterial cell lysis. I find that my protein goes in through the flow through fraction and I can't get any protein in the elution fraction. I incubated the cell lysate in the spin column at 4C in an end-to-end rotator for half an hour before spinning it around 1000 rpm to remove the unbound fraction. I add protease inhibitors and DTT to the cell lysate. I suspect that some of the glutathione beads are actually getting removed in the flow through fraction. Is it better to use Glutathione Sepharose beads instead of the GST- spin purification column?

[sify]

Yes even I use sepharose beads for purification of my protein. Although its time taking process. But protein loss in flow through is very less.

[biolcrazy]

sify, on 23 June 2011 - 12:29 AM, said:

Yes even I use sepharose beads for purification of my protein. Although its time taking process. But protein loss in flow through is very less.

thanks!
can you tell me which company's sepharose beads do  you use?

[mdfenko]

sepharose is the brand name of the agarose from ge healthcare (nee pharmacia).

Edited by mdfenko, 15 July 2011 - 07:10 AM.

[qzlabs]

biolcrazy, on 11 June 2011 - 10:00 AM, said:

I am using the Pierce GST spin purification column for purifying my GST tagged protein.  I have tried using the B-PER reagent/ sonication for bacterial cell lysis. I find that my protein goes in through the flow through fraction and I can't get any protein in the elution fraction. I incubated the cell lysate in the spin column at 4C in an end-to-end rotator for half an hour before spinning it around 1000 rpm to remove the unbound fraction. I add protease inhibitors and DTT to the cell lysate. I suspect that some of the glutathione beads are actually getting removed in the flow through fraction. Is it better to use Glutathione Sepharose beads instead of the GST- spin purification column?

What does it meaning By "incubated cell lysis in the spin column in an end-to-end rotator"? Does the lysate mix with the resin in the column? Usually, a spin column is prepacked.

[biolcrazy]

mdfenko, on 15 July 2011 - 07:09 AM, said:

sepharose is the brand name of the agarose from ge healthcare (nee pharmacia).

Thanks! i jus figured that out!

[biolcrazy]

qzlabs, on 15 July 2011 - 03:09 PM, said:

biolcrazy, on 11 June 2011 - 10:00 AM, said:

I am using the Pierce GST spin purification column for purifying my GST tagged protein.  I have tried using the B-PER reagent/ sonication for bacterial cell lysis. I find that my protein goes in through the flow through fraction and I can't get any protein in the elution fraction. I incubated the cell lysate in the spin column at 4C in an end-to-end rotator for half an hour before spinning it around 1000 rpm to remove the unbound fraction. I add protease inhibitors and DTT to the cell lysate. I suspect that some of the glutathione beads are actually getting removed in the flow through fraction. Is it better to use Glutathione Sepharose beads instead of the GST- spin purification column?

What does it meaning By "incubated cell lysis in the spin column in an end-to-end rotator"? Does the lysate mix with the resin in the column? Usually, a spin column is prepacked.

Yes the column is prepacked. The protocol says that the lysate should be added to the column and incubated in an end-to-end rotator. However the resin gets dislodged in the subsequent centrifugation steps and  it becomes difficult as the resin gets stuck in the middle of the column. When I just add more wash buffer and centrifuge to make the resin settle down I think some of glutathione beads get removed in the flow through and hence I lose protein in the flow through. I have tried avoiding incubating in the rotator and just let the lysate pass through (gravity flow) but lysate does not pass through and gets stuck. As soon as I centrifuge the column the resin gets dislodged.