Hi Andy,
There are lots of things to try. Here are a few:
Check your protein heterodimer combined pI, make sure you're at
least 1 pH unit away from that for your dialysis step.
Perhaps your dimer needs high NaCl to maintain its solubility -
try dialysing into a range of NaCl concentrations (100mM, 150,
200, 300 mM). Does it need any divalent cations? Check homologs -
do they bind divalent cations - try screening these in your
dialysis condition.
You say 2 column purifications - St - is that Strep affinity? Do
you have a lot of protein in the flow through from this column?
I've found that using pH 8 is very important for binding at this
stage - and high protein concentration.
I think the BB might be more helpful with a bit more information
about your expression method and if you think the protein is lost
at some stage during the purification, or only lost in aggregate
after dialysis/during concentration.
Good luck,
Fiona
On 30/01/2014 13:17, Anindito Sen wrote:
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Dear All,
This may be slightly off-the-track question but your feedback
will be very much appreciated. The situation is-
I obtain a very low amount of the protein of my interest (a
hetro-dimer) from the construct I am using (only 8% of the total
amount of protein obtained is the protein of my interest).
After 2 column purifications (Ni-NTA and St) the concentration
of the protein is around 0.24 mg/ml (volume- ~1.0 ml) from a
litre of bacterial culture and in ~300 mM NaCl present in the
elution buffer.
To reduce the high amount of salt I have I use a desalting
column which, further lowers the protein concentration
significantly.
I need atleast 1.0mg/ml of protein concentration and to the
amount of ~200 microlts for further experiments.
As the last resort I try to use high amount of bacterial
culture (~6lts) to scale up the yield and use centricon to
concentrate the protein at various stages. I am partially
successful to obtain 0.56mg/ml of protein concentration and up
to 50 microlts of it.
Another problem is that the protein is notoriously prone to
aggregation (> 1.5 mg/ml), so dialysis for ~ 2 hrs or so to
reduce the high salt concentration has failed miserably.
Please do send your feedback.
Thanks and Best Wishes
Andy
Dr. Anindito Sen (Ph.D)
Department of Cell
Biology & Anatomy
Graduate School of
Medicine
University of Tokyo
Tel & fax:
+81-3-5841-3339
--
Dr Fiona Whelan
Potts Group, L1
Department of Biology
The University of York
Phone: +44 (0) 1904 328673