Hi Nuria,

can you be more specific about the composition of your buffers?  If they are just Tris - NaCl (i.e. 20mM Tris HCl pH x.x ; 20mM Tris HCl pHx.x + 1M NaCl) or something similar, this should not be a problem, but could be altered to promote binding of the proteins you are looking for.

For me the Egg White may be a bigger issue as the viscosity could easily result in the column getting difficult to push in the same way that bacterial lysates containing intact genomic DNA can clog a column relatively quickly.  Can the source material be diluted, sonicated, filtered, separated by centrifugation etc to reduce the viscosity without destroying your target molecules?

Secondly - could you as an alternative use batch binding of loose Q resin instead of directly working with the column?

Strang's work in Glasgow suggests that dilution of the egg white into a buffer of extreme pH to isolate specific proteins coupled with coarse filtration through muslin to break down the viscosity before binding to resin in batch may be an approach that you could trial.  (search for the following reference).

PURIFICATION OF EGG-WHITE LYSOZYME BY ION-EXCHANGE CHROMA TOGRAPHY

R H C STRANG

Department of Biochemistry University of Glasgow Glasgow G 12 8QQ, UK ; Biochemical Education 12(2), 1984

best of luck with your experiments.

Hi Robin,

first of all, thank you for your quick answer and your suggestions.

Buffer A contains Tris-HCl 0,5M pH 7,2 and Tris-Base 0,5M Ph 7,2.

Buffer B contains exactly the same and NaCl 0,3M, also pH 7,2.

I obtained the egg white from ecological eggs separated from the yolk by hand, after that, the egg white was freeze-dried. Normally, I dilute 50mg of egg white in 10 mililiter of Buffer A, after that I filter this solution through 45 micrometer pore, and finally, I inject 1ml in the system.

Another thing that worry me is that, althoug I always load the same quantity of egg white and at the same concentration, I've never get two similar chromatograms (same peaks) at the end of the method, even though I wash and equilibrate the column after 4 or 5 runs.

Thank you again, and sorry for my poor english!

Hi Nuria,

Are you quite sure that your buffer composition is 0.5M of the buffering agent Tris?  This seems very concentrated - approximately 5-10 fold higher than any buffer I would use for a similar column, and this could also contribute to the general "thick-ness" of the liquid going through the column (in the same way that filtering 5M NaCl is much more difficult than 0.5M NaCl).

I am also curious about the use of both Tris-HCl and Tris base in the same buffer - this could equate to a final concentration of 1M Tris?  My understanding was that Tris base is the stock chemical, which you can then pH once dissolved using any one of a number of different acids, giving you Tris-HCl, Tris Acetate, Tris Borate etc.  Alternatively one can buy Tris HCl powder and dissolve this directly (but this only allows one to use HCl for pH).  Conventionally if I was making buffers with the same chemistry to those you have described, it would comprise simple Tris base at maybe 20mM / 50mM / 100mM concentration and the desired amount of NaCl, all pH equilibrated to 7.2 using HCl : but no higher concentration of Tris than this final.

Now that you have described how your egg white is prepared, I do not think this is the problem for the viscosity : it is a small amount of freeze-dried protein, 0.45µ filtered, and should not be the problem.

So perhaps your initial thought that your buffers were the problem is correct.

I hope that this does prove to be the answer, and wish you the very best for your experiment.  Feliz Navidad y bueno ano nuevo!  (ps, your English is much better than my Spanish!!)

Not only the buffer concentration is very high, and may prevent protein binding to an IEC column. Also, protein at 5 mg/ml will increase viscosity. Note that in IEC (unlike gel filtration) sample volume can be quite large.

1. Your description of the Tris buffer is imprecise. What is the final concentration of Tris in the buffer whether derived from Tris HCl or Tris base.  For a buffer that has 0.5M Tris in total with a counter ion of chloride  and a pH of 7.2 then this is properly described as 

0.5M TrisHCl buffer pH 7.2. Is this what you are using. 

2. As indicated by the previous answers the concentration of Tris and NaCl are very high  for allowing the protein the stick to the column. However if  the albumin is sticking to the column and you are loading a large  quantity then the column may bind so much that it begins to clog up the column as effectively as if it had precipitated and the flow will be reduced rapidly . 

Try using a lower concentration of Tris as suggested in previous answers and try loading much less albumin.

You can easily see if the buffers are to blame by running the bind/wash/elution program without sample. I've never seen significant back pressure from simple buffers before, only from samples, but the simple test above will tell you the answer. Quite separately your Tris concentration seems far too high (10mM or 20mM or 50mM would be more usual, with a gradient of NaCl from 0 to 1M). Finally Tris is not a great buffer at pH 7.2 as its pKa is 8.1. I'd be inclined to raise the pH if you wish to use Tris.  

You don't mention the size of the column, so it is difficult to know if the amount of protein is resonable. I would never use a small bead such as Mono-beads as a first step with a crude preparation. Capto or Separose FF would be my choise. I assume that the 0.5M concentrations are your untitrated stocks of Tris-base and Tris-HCl that are mixed to obtain the pH you want. As mentioned above pH 7.2 is on the edge of what is OK for Tris. HEPES would be a buffer you could try instead.

 The Egg White material is the cause of your increased pressure. You should look into possible fractionation by Ammonium sulfate or other means to clean up the egg white protein before doing any Ion Exchange chromatography. Also I would tend to move away from the use of Tris and use Sodium Phosphate buffer system for the 

First of all, Happy New Year!!

I made a mistake, the real concentration of my buffers is 50mM with a gradient of 30mM of NaCl. The size of the column is 1ml, I've washed it several times as indicated in the instructions of the column, also, I washed the column leaving it overnight in a solution of 1mg/ml pepsin, 10mM acetic acid and 50mM NaCl.

After all of these methods, the column operates correctly during 3 or 4 runs, but, I've never get the same chromatogram, so I can't obtain the separated proteins.

Thank you all for your advices!!!!!