Do you really need to remove the NaCl? Some ionic strength is often
necessary to stabilize proteins. Our routine purification buffers all
contain at least 100 mM NaCl. This will not usually interfere with
crystallization screening.
To minimize the probability of aggregation, you need to (1) ensure that
the buffer pH is not close to the pI--pH 8.00 is a safe choice for many
proteins, (2) probably maintain 100-150 mM ionic strength, and (3)
consider solubility-increasing additives, like glycerol. However,
solubility additives are going to seriously interfere with
crystallization, but sometimes you have no choice.
Bear in mind that centrifugal concentrators may bind protein. This may
become noticeable when using small absolute amounts of protein. Usually
PES (polyethylenesulfone) concentrator membranes have the least protein
adsorption.
Good luck!
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 1/30/2014 8:17 AM, Anindito Sen wrote:
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*
