[ccp4bb] Troubleshooting protein purification cation IEX

- Protein crystallography

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- Crystallisation

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Troubleshooting protein purification cation IEX
From: Meg mgbiotec {- at -} GMAIL {- dot -} COM
Date: 2008-09-23

Dear All,

This is with reference to the purification of our recombinant protein sample
expressed in E.coli as inclusion bodies. After Solubilization refolding we perform
the cation exchange chromatography of our protein sample using SP
sepharose fast flow resin. Attached herewith find the SDS PAGE and IEF
results of the collected fractions.

In addition to our protein of interest we are also getting high molecular weigh
contaminants, which we cannot get rid of in IEX. Can anyone please guide me
on a technique to get rid of these bands as even after gel filtration of samples
few high mol wt contaminant bands are not separated from main proteins and
sample gets diluted too.

In cation IEX procedure is
Column Sp Sepharose Fast flow packed in fineline 35 column packed bed
volume 100 ml
System AKTA FPLC
Equilibration 10 C.V. protein loading 3-4 C.V. [approx 1mg/ml protein conc],
washing to remove unbound materials 2 C.V. step elution 0-35% gradient – 1
C.V., 35 –80% gradient – 10 C.V. and 60 –100% gradient 1 C.V.
Protein elutes at 40-50% gradient.
Protein details: Our protein is stable at acidic pH and has a pI of 5.8 –6.3 and
buffer is Na Acetate buffer pH 4.5 and elution buffer is starting buffer
containing 0.4 M NaCl.

We get only one peak on AKTA but on running SDS page we get so many
bands even IEF shows 1-2 bands at the most.

How can we modify the method or what can be done to get rid of extra high
mol wt bands.

Any help will be deeply appreciated.

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Off topic: GPU accelerated crystallographic calculations
From: Pete Meyer pameyer {- at -} MCW {- dot -} EDU
Date: 2008-09-23

Next message:
Subject: Question: Linux distributions people use
From: "Winter, G (Graeme)" g {- dot -} winter {- at -} DL {- dot -} AC {- dot -} UK
Date: 2008-09-23