Desalting columns

From: Sangeetha Vedula
Date: 27 February 2012 16:01


Dear bb users,

I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL.

I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums.

Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column.

Thanks a lot!

Best regards,

Sangeetha.

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From: Tim Gruene 

Dear Sangeetha,

provided you express the protein in E. coli, you could also sonicate the
cell debris in various buffers and compare supernatant/pellet on
SDS-gel. It might already give you a first clue and is faster, cheaper
and you don't risk to clog the new columns with aggregated protein.

Tim

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From: Dima Klenchin

I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL.

I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums.

Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column.

~ 1 ml spin columns with Sephadex G25 or Biogel P6. Typically, when loading 200 ul of the sample onto 1 ml column, desalting efficiency is ~ 95% and protein recovery is ~ 90-95% without volume change. Lowering sample volume will increasing dealting and reduce yield but I never had the yeild worse than 75% even with 50 ul volume.

Bio-Rad sells prepacked "Bio-Spin P-6" or you can buy empty columns and dry P6 (by "fine" grade in this case) and pack to 1.2 ml per column, which will take your 250 ul volume and still result in decent desalting. Pharmacia probably sells something similar and many companies sell 0.5 ml spin columns.  I like P6 (polyacrylamide) better than Sephadex (dextran) because usually (but not always) non-specific binding is lower.

- Dima

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From: Bernhard Rupp (Hofkristallrat a.D.)

Why, in the first place, do you feel an urge  to concentrate your protein above 3 mg/ml ?

 

For crystallization, the concentration needs to be

a)      high enough to achieve supersaturation, meaning close enough to the maximum solubility in a given buffer so that the precipitant can drive the system in to supersaturation, preferably of a level where homogenous nucleation can occur (or you micro-seed, if necessary)

b)      high enough that sufficient material for crystals of acceptable size to grow is in the drop, which is generally the case, lest micro-crystal showers happen.

 

There is ample evidence for proteins crystallizing below 3 mg/ml.

 

The often quoted PDB/BMCD average of somewhere around 10 mg/ml is biased towards highly soluble, smaller (lower hanging fruit) proteins.

 

Sometimes the shape of a distribution matters ;-)

 

BR    

 

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From: Xiaodi Yu

Hi Sangeetha:

If you just want to check which buffer is good for your protein, maybe you can try to set up a crystallization screen, keeping your protein concentration just 3 mg/ml. You can observe (after several days) which conditions give you a clear drop, and maybe you can find a clue which buffer is better for your protein. If you want to speed up the whole processes, you can also add glycerol into the drops to grasp the water molecules.

Yu Xiaodi

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From: Patrick Shaw Stewart

We did some data mining from remark 280 of the PDB in 2004.  Of the 1000 entries that listed [protein], 46 proteins were crystallized below 3.1 mg/ml.  See table 3 at  http://www.douglas.co.uk/PDB_data.htm .

Patrick

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From: Andre Luis Berteli Ambrosio


Hi,
We have a case where a 260 KDa protein crystallized at 3 mg/ml, that was the highest concentration we could achieve, without playing with the protein buffer.
Protein-to-well-solution ratio was 3:1.
When screening, we tested all ratios between 4:1 and 1:1. Crystals only appeared at 3:1 ratio.
That is another important variable to test, I guess.
Regards,
-Andre.

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From: Bernhard Rupp (Hofkristallrat a.D.)

Ø   in 2004.  Of the 1000 entries that listed [protein], 46 proteins were crystallized below 3.1 mg/ml.  

 

That is not necessarily the success rate for low concentrations, which we actually would like to have. We would need negatives for < 3 for to give a correct answer. I guess even occurrence might be more frequent now. <hint>Something for our center data miners who have the negatives kept </hint>.

Anecdotally, my personal below-3-occurance <brag> not success, that is 100% </brag> is ~15%, but that does not mean much.

Even if it were the success rate:  5% chance compared to zero chance when precipitated – I'd take it and probably learn something useful during the experiment.

BR  

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From: Christian Roth


If you want test a lot of different conditions a Thermofluorecence assay might
work for your protein and you may find a condition which stabilise your
protein. However there is no warranty that it crystallise better afterwards.

Christian

Am Montag 27 Februar 2012 17:01:33 schrieb Sangeetha Vedula:

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From: Prince, D Bryan


Actually, I would refer the ccp4-bbs to Journal of Structural Biology
175 (2011) pp216-223 for the use of fluorescence in relation to protein
crystallization.

Regards,
Bryan