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Protein is not Binding to DE52 column material

DE52 Binding of protein

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#1 Monita

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Posted 10 April 2013 - 02:08 AM

Dear All,

I'm trying to purify a protein (pI=6.95) using DE52 column material. The buffers that I'am using to purify are Buffer A: 20mM Tris - Cl, pH 8 + 1mM EDTA; Buffer B: 20mM Tris - Cl, pH 8 + 1mM EDTA + 1M NaCl (prepared at room temperature). The purification is carried out at 4 degree C. I start with equilibrating the column material with 0.5M Tris-Cl, pH 8 buffer and once the pH reaches 8 I switch to eqilibrating with the Buffer A. I dialyze the sample against buffer A before loading to the column. Every time, the protein seems to elute in the load and wash flow through. I am not able to understand why the protein is not binding to the column. Kindly help.

Regards,
Monita

#2 mdfenko

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Posted 10 April 2013 - 06:44 AM

some possibilities:

are you certain none bound to the matrix? you may have exceeded the capacity.

the ionic strength of your starting buffer may be too high, pH may be too low.

equilibration may not be complete.

is the de52 fresh? if not, have you cycled (regenerated) it (acid, water, base, water, then buffer)?

whatman (now ge healthcare) used to have a handbook available. i can't find it online (i may have a printed version someplace). i am attaching an older version of ge healthcare's ion exchange handbook. it contains information on deae-sephacel, which is similar to de52. i hope it helps.

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#3 Monita

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Posted 15 April 2013 - 01:12 AM

Dear mdfenco,


Yes, none bound to the matrix because I ran a gel to see if the protein of interest is present in any of the fractions. I did not see a band at that particular molecular weight region. At the start of the experiment there is no NaCl added and the buffer used is 20 mM Tris -Cl buffer pH 8. Each time I tried to purify the protein I have made sure to use fresh material. That now narrows my problem to equilibration. Thanks for the ebook :).

Regards,
Monita




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