GST-tag protein purification - GST-tag protein purification (May/05/2009 )
I'm trying to purify the GST-tag protein and I use the GST affinity column but it's too slow.
What can I do to let it be more faster or other ways to do this??
Second problem is when I done purification and run the SDS-PAGE, I found some protein expressed I don't want.
Whether the protein degradation when I purification or it already degradation before I purification the protein??
-breezedemon-
Hey, You can use a pump, FPLC to speed it up. DNA precipitation would make teh lysate less viscous and the column will run faster.
You need to give more information.
It could be some stop codon that got introduced or some protease or yr purification. Chk for all, chk yr clone first, use chilled buffers and protease inhibitors during purification.
Best,
TC
breezedemon on May 5 2009, 06:38 PM said:
I'm trying to purify the GST-tag protein and I use the GST affinity column but it's too slow.
What can I do to let it be more faster or other ways to do this??
Second problem is when I done purification and run the SDS-PAGE, I found some protein expressed I don't want.
Whether the protein degradation when I purification or it already degradation before I purification the protein??
What can I do to let it be more faster or other ways to do this??
Second problem is when I done purification and run the SDS-PAGE, I found some protein expressed I don't want.
Whether the protein degradation when I purification or it already degradation before I purification the protein??
-T C-
breezedemon on May 5 2009, 02:08 PM said:
I'm trying to purify the GST-tag protein and I use the GST affinity column but it's too slow.
What can I do to let it be more faster or other ways to do this??
Second problem is when I done purification and run the SDS-PAGE, I found some protein expressed I don't want.
Whether the protein degradation when I purification or it already degradation before I purification the protein??
What can I do to let it be more faster or other ways to do this??
Second problem is when I done purification and run the SDS-PAGE, I found some protein expressed I don't want.
Whether the protein degradation when I purification or it already degradation before I purification the protein??
I assume that you mean the GSH-column runs slowly. This is most probably due to DNA in the lysate. A simple method is to batch bind.
Incubated your lysate in a 10 ml tube with the GSH-sepharose for 1 hour at 4 oC on a rotator. Then wash the resin x3 with buffer (by gentle centrifugation/resuspension). Pack into a column, wash until A280 is baseline then elute as normal.
As for the protein you "donīt want". Are you sure that it is a degraded version of the recombinant protein (eg reactive in a western) and not just a contaminant from the expression host? Column chromatography of thick lysates almost always gives impure product. Try batch binding.
If you have degradation of the recombinant protein, prepare the lysate in the presence of a cocktail of protease inhibitors and keep cold.
Only worry about things like premature termination and in vivo degradation when you are SURE it is not a problem with the lysis/chromatography steps.
Hope this helps.
-klinmed-
T C on May 5 2009, 04:10 PM said:
Hey, You can use a pump, FPLC to speed it up. DNA precipitation would make teh lysate less viscous and the column will run faster.
You need to give more information.
It could be some stop codon that got introduced or some protease or yr purification. Chk for all, chk yr clone first, use chilled buffers and protease inhibitors during purification.
Best,
TC
You need to give more information.
It could be some stop codon that got introduced or some protease or yr purification. Chk for all, chk yr clone first, use chilled buffers and protease inhibitors during purification.
Best,
TC
breezedemon on May 5 2009, 06:38 PM said:
I'm trying to purify the GST-tag protein and I use the GST affinity column but it's too slow.
What can I do to let it be more faster or other ways to do this??
Second problem is when I done purification and run the SDS-PAGE, I found some protein expressed I don't want.
Whether the protein degradation when I purification or it already degradation before I purification the protein??
What can I do to let it be more faster or other ways to do this??
Second problem is when I done purification and run the SDS-PAGE, I found some protein expressed I don't want.
Whether the protein degradation when I purification or it already degradation before I purification the protein??
The interaction between GST and GSH is slow so don't try and speed up the flow rate otherwise your protein won't bind (max = 0.5ml/min, I run mine at 0.2ml/min)
Batch purification is quicker but often co-purifies contaminants so it depends on your downstream application. If you don't need a perfectly pure prep do the batch purif, otherwise be patient and run the column.
I agree that it sounds like you have degradation, use protease inhibitors and keep everything at 4C
P
-Penguin-
breezedemon on May 5 2009, 05:08 AM said:
I'm trying to purify the GST-tag protein and I use the GST affinity column but it's too slow.
What can I do to let it be more faster or other ways to do this??
Second problem is when I done purification and run the SDS-PAGE, I found some protein expressed I don't want.
Whether the protein degradation when I purification or it already degradation before I purification the protein??
What can I do to let it be more faster or other ways to do this??
Second problem is when I done purification and run the SDS-PAGE, I found some protein expressed I don't want.
Whether the protein degradation when I purification or it already degradation before I purification the protein??
Hi
GST Fusion proteins are easily bound on to the GST sepharose FF resins(GE). I used to run the sample at 90cm/hr which i used to get more than 85% binding of the GST fusion protein. Your sample should be clear (0.45 micron filtered) before applying on the GST Resin then it will go easily through the column. Optimizing the washing conditions for chromatography will give pure protein. u can detect the degraded proteins by running on to a western blot and detecting with Anti GST antibody or your specific protein Antibody.
-sudhakar mutyala-