I'm trying to purify a GST tagged protein. We see expression in the lysate, sonicate, but when we incubate with GST beads, we lose everything. We see non specific proteins in the washes, but by elution time no proteins at all are left. We are incubating ON at 4 C, without protease inhibitors. However, we should not see such complete protein degradation. Any suggestions about where we are losing the protein?
Thanks!
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#2
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an elder
Posted 05 November 2009 - 10:58 AM
are you sure it's not still bound to the beads?
you may need to adjust your elution conditions.
are you sure it's not in the wash?
you may need to adjust your binding conditions.
you may need to adjust your elution conditions.
are you sure it's not in the wash?
you may need to adjust your binding conditions.
talent does what it can
genius does what it must
i do what i get paid to do
genius does what it must
i do what i get paid to do
#3
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Posted 05 November 2009 - 12:00 PM
mdfenko, on Nov 5 2009, 10:58 AM, said:
are you sure it's not still bound to the beads?
you may need to adjust your elution conditions.
are you sure it's not in the wash?
you may need to adjust your binding conditions.
you may need to adjust your elution conditions.
are you sure it's not in the wash?
you may need to adjust your binding conditions.
We ran wash, beads, pellet, and the kitchen sink on gels and we loose somewhere after sonication. I've tried a couple of different binding conditions, but when I run the lysate on a gel after bead incubation it's gone, so presumably bound to the beads.
#4
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an elder
Posted 06 November 2009 - 09:00 AM
you ran the beads on the gel and it didn't show up?
are you sure the protein was in the solution you loaded (why did you sonicate after lysis? is it a membrane bound protein?)?
overnight at 4C can allow enough protease activity to degrade your protein , especially if it is particularly susceptible to proteolysis. you should add inhibitors.
are you sure the protein was in the solution you loaded (why did you sonicate after lysis? is it a membrane bound protein?)?
overnight at 4C can allow enough protease activity to degrade your protein , especially if it is particularly susceptible to proteolysis. you should add inhibitors.
talent does what it can
genius does what it must
i do what i get paid to do
genius does what it must
i do what i get paid to do
#5
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Posted 13 November 2009 - 10:53 AM
momboys3, on Nov 6 2009, 01:24 AM, said:
I'm trying to purify a GST tagged protein. We see expression in the lysate, sonicate, but when we incubate with GST beads, we lose everything. We see non specific proteins in the washes, but by elution time no proteins at all are left. We are incubating ON at 4 C, without protease inhibitors. However, we should not see such complete protein degradation. Any suggestions about where we are losing the protein?
Thanks!
Thanks!
hi.... is that protein is coming is soluble form? i think most of your protein is getting in inclusion body...incubate your culture at 18c O/N...... then proceeds further....also add protease inhibitors through out.
all d best
