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Proteins sticking to Dialysis Bag


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#1 anonymous

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Posted 28 June 2001 - 09:00 PM

My proteins stick to the dialysis tubing.
I use Sigma brand 6-8 KDa cutoff tubing, pre-washed in hot, then cold water, then double distilled water. I dialyse against 5 litres of ddH2O over 24-48 hours, with at least 6 changes of water, performed at 4 degrees Celsius.

I have tried using Tris buffer 25mM and also phosphate-buffered saline, but they both precipitate at 4 degrees.

I stained the used dialysis bag with Coomassie Blue which showed the protein quite obviously. The unused bag doesnot stain blue.

The proteins are those extracted from human urine or crystals derived from human urine. They include prothrombin (and its fragments), osteopontin, human serum albumin, tamm-horsfall glycoprotein, and many others.

#2 anonymous

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Posted 28 June 2001 - 09:00 PM

You don't explain why you're dialysing. Can you try different pH buffers or including various concentrations of salt? If the protein is selectively sticking to the dialysis membrane (as opposed to simply precipitating out, you could try buffer exchanging using Sephadex G25 (e.g. PD10 column for small scale).
Jim

#3 anonymous

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Posted 29 June 2001 - 09:00 PM

I am dialysing to remove the salts from the urine as well as the EDTA, Ca and Oxalate from my demineralisedCalcium Oxalate crystals.

Any ideas on how to FLUSH the dialysis bag to remove the proteins?

For small scale I use a P6 DG desalting column, but for large scale need to dialyse.


#4 anonymous

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Posted 10 July 2001 - 09:00 PM

How do you prepare the dialysis tubing? We generally boil the tubingin a 2% solution of NaHCO3 for 5 minutes and then rinse the tubing with MilliQ/D.I water before adding the protein sample to it.




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