Hi Nazia,
Answer to your last email about handling the detergents. It is suggested
for all the detergent powders to be stored at -20 in a desiccator box or
simply the bottle should be properly sealed with parafilm since the
detergent powders are very hygroscopic and quickly absorb moisture. There
can be two ways to use detergent. First, take the detergent out of -20 and
keep at room temperature for sometime until the bottle comes at the room
temperature, thereafter you can weigh them. If you don't do this, every
time your detergent will absorb some moisture and will change chemically.
The other option is to make 10-20 % (w/v) solution of the detergent in
water, filter it, make 1-2 ml aliquots and store them at -20. Avoid freeze
and thawing.
And it seems like there is some problem in your Akta because your buffer is
completely fine and I have used the similar buffer even with 10 % glycerol.
Vikrant
On Sat, Jul 27, 2013 at 2:12 AM, Nazia Nasir Phd2009,ProteinCrystall.Lab <
nazia.nasi...@nii.ac.in> wrote:
> Dear all,
>
> Thank you for the prompt and valuable suggestions. I had made a typo in my
> earlier mail. I use 50mM NaCl, not 5mM NaCl. I am facing this problem only
> while using DDM in buffers. My other runs, even with 8M urea run perfectly
> fine.
>
> I will try increasing the salt concentartion and degas by methods
> suggested.
>
> All the detergents I use are from Anatrace and store them in a vacuum box
> at -20. But just befre weighing, I don't bring the detergent to room
> temperature before weighing. Would this be causing the problem, as
> suggested by Bert? If yes, how do you handle the detergents?
>
> Thanks again!
>
> -Nazia
>
>
>
>
> On Sat, Jul 27, 2013 at 6:09 AM, Ho Leung Ng wrote:
>
>> If your buffer can't go through a 0.2 micron filter easily, you
>> shouldn't run it through your FPLC. Detergent purity may be an issue.
>> I also experienced problems filtering a buffer containing CHAPS from
>> vendor X. When I switched to Anatrace CHAPS, no more clogging.
>>
>>
>> Ho
>>
>> Ho Leung Ng
>> University of Hawaii at Manoa
>> Assistant Professor, Department of Chemistry
>> h...@hawaii.edu
>>
>>
>>
>> We are trying to purify a membrane protein using different detergents
>> (DDM,
>> OG etc.). We have tried using 1mM DDM in 20mM Tris, 5mM NaCl and 5%
>> glycerol buffer to purify the protein. however, we are facing problems in
>> running the buffer in 16/60 Superdex 200 pg gel filytration coloumn using
>> AKTA Explorer. The entire machine is in a cold cabinet. The buffer was
>> also
>> kept at constant slow stirring, thinking that it might be getting
>> precipitated, which we are not able to see. Still the back pressure is
>> very
>> high and the in-line filter keeps clogging. We have filtered the buffer
>> through a 0.2 micron filter, which too was very difficult. the
>>
>> Has anyone faced a similar proble? Or is there a way that buffers with
>> detergents are supposed to be made? Or are there any particular coloumns
>> meant for such runs.
>>
>
>
>
> --
> Nazia Nasir
> PhD Scholar
> Protein Crystallography Lab
> National Institute of Immunology
> New Delhi
>
