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Couple questions about GST purification - (Aug/06/2009 )

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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?

-Niraj-

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

-swanny-

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% Triton X-100, 50 g of DNase I, 30 g of PMSF per ml, 1 g of aprotinin per ml 1 g of pepstatin A per ml, 10 g of leupeptin per ml as per
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

-Niraj-

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% Triton X-100, 50 g of DNase I, 30 g of PMSF per ml, 1 g of aprotinin per ml 1 g of pepstatin A per ml, 10 g of leupeptin per ml as per
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).

-swanny-

Thanks a lot Swanny...

-Niraj-


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.

Hi Swanny,
I had used the above buffer for downstream washing of complex. And it is working great.
Thanks a lot.
Niraj

-Niraj-

Excellent news! Glad I could help.

-swanny-
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