16 replies to this topic

[p3t3r1]

Hey guys, I have a couple of questions about the GST purification of my protein (~50 kD)

1. When I elute my protein off the Gluthione Sephrose 4B column, I also get Hsp60 protein that binds with it. Is there a way to get rid of the Hsp60? I tried 0.1 % tritonx + DTT and 1% triton-x + DTT in my washing buffer for the column but the hsp60 doesn't seem to come off.

2. There seems to be significant degradation of my protein after expression and doing subsequent purification attempts. I did everything I could to prevent it. I used Roche's protease inhibitor cocktail in my lysis buffer, my wash buffer, and even on my beads. I also added 1mM PMSF and kept everything perfectly cold ( I work in the cold room).

Any suggestions?

Thanks alot!

[Roo]

Quote

2. There seems to be significant degradation of my protein after expression and doing subsequent purification attempts. I did everything I could to prevent it. I used Roche's protease inhibitor cocktail in my lysis buffer, my wash buffer, and even on my beads. I also added 1mM PMSF and kept everything perfectly cold ( I work in the cold room).

I'm not sure about your first question, but it's possible that your degradation is occurring during the induction so it may be too late by the time you get to the lysis/purfication steps.  You may have to try different growth/induction conditions like inducing at 30 degrees or room temp instead of 37 degrees.  You might also want to try varying the IPTG concentration.  If none of this works, then you can also try cloning into a different GSTvector (or switching to His, MBP, etc), but obviously that will take a little more time and effort.

[p3t3r1]

Thank you for your help but my induction conditons are already *optimized*- 19 hrs at room temperature. This seems to give me the largest yield of protein. Maybe I should change  the # of hrs but keep temp the same? Tried 37 C before but failed badly, 30 C is known to not work too well with similar proteins.

[Roo]

19 hours seems really long for an induction, but I've only used 3 - 6 for mine.  It sounds like you have been working on varying some conditions, but remember that just because you get a large yield at 19 hours (or whatever time) doesn't mean you will get "good" protein.  Definitely adjust the induction time.  You may get less protein, but if less of it is degraded then it is definitely worth the decrease in the overall yield.

[T C]

Hey,

Try using 500 mM NaCl in the wash buffer and wash extensively.  Also, did you try FPLC?

Best,
TC

[quote name='p3t3r1' date='Aug 6 2009, 10:15 PM' post='32035'

1. When I elute my protein off the Gluthione Sephrose 4B column, I also get Hsp60 protein that binds with it. Is there a way to get rid of the Hsp60? I tried 0.1 % tritonx + DTT and 1% triton-x + DTT in my washing buffer for the column but the hsp60 doesn't seem to come off.

[/quote]

[archit]

1. Before elution give washes with increasing NaCl stringency such as 100, 200, 300 mM NaCl, and then elute .collect samples at each step check for loss of your protein and for non specific bound proteins. after getting a good elution which corresponds to your protein of interest without contamination confirm with a western blot.  :)

[archit]

2. Do a western analysis loading the induced cell pellet and your eluted sample, you will come to know whether it is degradation during the induction or your protein is not stable. if you are extra careful and work in the cold room completely, and are sure that the protein is still degrading then check whether this protein requires some binding partners for stability. this other protein might have to be co-expressed. Otherwise you can also try induction in the presence of a specific inhibitor of your protein so that it is not active during the overexpression period and might not get degraded. you might have to play around with the standardization for both.
best, archit.

[swanny]

We always added 10% glycerol to our wash solutions to get rid of non-specific binding.

[nirajm]

swanny, on Aug 16 2009, 04:20 PM, said:

We always added 10% glycerol to our wash solutions to get rid of non-specific binding.

What is the recipe of the wash solution you used?

Edited by nirajm, 20 August 2009 - 10:26 AM.

[swanny]

nirajm, on Aug 21 2009, 04:25 AM, said:

swanny, on Aug 16 2009, 04:20 PM, said:

We always added 10% glycerol to our wash solutions to get rid of non-specific binding.

What is the recipe of the wash solution you used?

Your normal lysis solution plus 10% glycerol. The ancient text known as my PhD thesis used this recipe:
100 mM Tris, pH7.5, 150 mM NaCl, 0.1 mM PMSF, 2% Triton X-100, 0.1% mercaptoethanol, 10% glycerol.

[nirajm]

swanny, on Aug 20 2009, 09:30 PM, said:

nirajm, on Aug 21 2009, 04:25 AM, said:

swanny, on Aug 16 2009, 04:20 PM, said:

We always added 10% glycerol to our wash solutions to get rid of non-specific binding.

What is the recipe of the wash solution you used?

Your normal lysis solution plus 10% glycerol. The ancient text known as my PhD thesis used this recipe:
100 mM Tris, pH7.5, 150 mM NaCl, 0.1 mM PMSF, 2% Triton X-100, 0.1% mercaptoethanol, 10% glycerol.

Thanks a lot and I ll try this. I am doing 3 washes of 10 m each. Is it ok?

[swanny]

Washing with 10 - 20 column volumes will be fine, or you can just wash until the UV trace returns to baseline.

[nirajm]

Hi Swanny,
I am washing the protein complexes with GST binding buffer.
540 mM NaCl, 2.7 mM KCl, 10.15 mM Na2HPO4, 1.75 mM KH2PO4, 10 mM MgCl2, 1% [vol/vol] Triton X-100, 50 µg of DNase I, 30 µg of PMSF per ml, 1 µg of aprotinin per ml 1 µg of pepstatin A per ml, 10 µg of leupeptin per ml [pH 7.4] as per [Journal of Virology, June 2004, p. 6459-6468, Vol. 78, No. 12]
So, I am thinking of using your lysis buffer at the time of washing.
What buffer you were using for binding?
Thanks a lot for your response.
Niraj

[swanny]

nirajm, on Aug 26 2009, 06:44 AM, said:

Hi Swanny,
I am washing the protein complexes with GST binding buffer.
540 mM NaCl, 2.7 mM KCl, 10.15 mM Na2HPO4, 1.75 mM KH2PO4, 10 mM MgCl2, 1% [vol/vol] Triton X-100, 50 µg of DNase I, 30 µg of PMSF per ml, 1 µg of aprotinin per ml 1 µg of pepstatin A per ml, 10 µg of leupeptin per ml [pH 7.4] as per [Journal of Virology, June 2004, p. 6459-6468, Vol. 78, No. 12]
So, I am thinking of using your lysis buffer at the time of washing.
What buffer you were using for binding?
Thanks a lot for your response.
Niraj

Binding was with lysis buffer (that is, no glycerol), washing was 10 cv lysis buffer plus glycerol.

In your situation, I'd add glycerol to your lysis buffer and wash with it. When using glycerol, I would go for a lower salt concentration. You can also exchange into another buffer for downstream steps (we were doing a thrombin cleavage, so we exchanged into it).

[nirajm]

Thanks a lot Swanny...