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chromatography question


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#1 Dawgology

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Posted 02 July 2013 - 10:55 AM

Hi all,
I have a chromatography issue and could use some advice. I am currently trying to purify a 196 KDa DNA binding protein. The protein binds well to either heparin or DEAE. The problem is getting it off the resin. More specifically getting it off all at one elution step. I am loading the columns at 50 mM NaCl and using a step gradient to elute. The protein begins to come off at around 200 mM. But then it won't even near all elut at that salt concentration. Most of it stays bound and then more will come off at 300mM, then more at 400mM and so on until it pretty much all finally gets bumped off somewhere over 500mM.
Any thoughts would be appreciated

#2 mdfenko

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Posted 03 July 2013 - 04:15 AM

either pool all the steps or eliminate the earlier steps and go to 500mM after washing.

if that's not pure enough then you could use a gradient and only pool the fractions that meet your purity requirements.
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#3 labtastic

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Posted 07 July 2013 - 02:45 PM

This is not unusual to have a protein elute over a wide salt concentration. My suggestion is to stop messing with multiple step washes and just use a gradient. You don't need a fancy FPLC to run a gradient if you are doing this by gravity. You can use a gradient maker, or, if you don't have one, just get some tubing and run a siphon from a beaker with high salt to a beaker with a stir bar on a stir plate containing the low salt buffer, and then a second siphon leading out of that beaker into your column. Decrease your buffer volumes to make a steeper gradient, which will minimize the volume your protein is eluted in, but doing this can compromise resolution. It may not matter though because it's not uncommon to require a second purification step (perhaps using your heparin resin, or gel filtration).

Edited by labtastic, 07 July 2013 - 02:47 PM.





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