Dear All,

1st of all , Thanks for the very quick feedback. I will answer your questions to make the picture more clear-

Nicolas-

Yes the dimer is co-expressed. Almost all the protein is in the sup and not in the pellet. Multiple-step  dialysis has failed. I have been trying various controls and methods for the last 2 years but of not great success. I do have significant amount of protein in the 1st step all including homo-dimers and the hetro-dimer. Only 8% of that amount is hetro-dimer. 

Fiona-

I will look in the pI option. Step dialysis has failed due to the aggregation issue. St is for Strep affinity. Yes, there is a lot of protein in the flow through from the column, I have checked and those protein are the homo-dimers. 

Roger-

I do need to remove/reduce the salt for cryo-Electron Microscopy (cryo-EM). The maximum that can be afforded is 50 mM. Glycerol is a big no for cryo-EM so I am not sure how to implement it. You are absolutely correct as the membranes of the centrifugal concentration devices are creating one big problem. I will try PES (polyethylenesulfone) concentrator. The final pH needs to be 6.8-7.0 at the for the EM work. So to make a drop from pH 8.0 to 6.8 will be a additive step in purification process. Can you suggest how can we do it without much drop in the concentration ?

Bryan-

I agree with you that the plasmid I have in my hand is far from being a good candidate for the purification process, so I have to venture more. Your point No2 & 3 is well taken, I will look into that. However I cannot have the pH as high as 8. It needs to be around 6.8 to 7.0 for EM work. 

Lastly I am not an expert of Protein Purification , my expertise are in Cryo-EM and computational  image processing, so I have to go baby-steps of asking such questions at the forum. Thanks again. 

Best 

Andy






Dr. Anindito Sen (Ph.D)
Assistant Professor (Project)
Department of Cell Biology & Anatomy
Graduate School of Medicine
University of Tokyo
Tel & fax: +81-3-5841-3339

On Jan 30, 2014, at 10:57 PM, Fiona Whelan <[log in to unmask]> wrote:

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:
[log in to unmask]" type="cite"> 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