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FPLC - why is it so hard to filter my crude extract?


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#1 Luria Bertani

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Posted 28 June 2012 - 01:13 AM

Hello,

I've been noticing recently that I can have a VERY difficult time filtering my crude extracts.
Basically, when I express my protein(s) in E.Coli I spin them down (5000 x g) then sonicate them (add in Lyzozyme, DNAse and MgCl2) and add protease inhibitor
and then spin down for 30 minutes at 18 000 x g. This gives a pellet (discard) and a supernatant.

Before loading into the FPLC, I have to syringe filter it through an 0.22 uM (micrometer) filter before loading onto the FPLC column (to prevent blockages).
Sometimes this is EXTREMELY HARD to do with the liquid resisting. It can be very frustrating at times - some solutions I have tried have been to spin
down a second time (1 hour x 18 000 x g) or dialyze overnight in buffer or add more DNAse and leave overnight. But often this does not help.

So what is going on? This doesn't happen to every batch of purification I do.

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#2 mdfenko

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Posted 28 June 2012 - 08:27 AM

the resistance is concentration and membrane type dependent.

higher solute concentrations will give greater resistance.

hydrophobic membranes will be very resistant regardless of concentration.
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