Hi,
Many things can lead to your observation. Please outline all steps of
your purification procedure as it is not clear what is done before
and after the Ion Exchange steps.
I am not sure if IEF in your emails refers to Isoelectric focusing,
as the acronym is usually used??
Couple of suggestions:
1) Instead of contamination, you might just be seeing multiple bands
due to 'aggregation' of your protein! Make sure you boil the sample
prior to loading on gel and also that your loading dye contains SDS,
bME/DTT.
2) We used to do entire purifications with inclusion body preps under
denaturing conditions to prevent unwanted aggregation of partially
folded or misfolded species. Not sure if denaturant is present all
along.
3) If the problem is of contamination, try making the gradient
shallow for the 35 –80% gradient step in Ion Exchange (increase to
say, 20 cv or more).
4) If the problem is of contamination, try to add more steps to
purification -- e.g., affinity step (if possible), anion exchange as
well etc.
5) If IEF (in the sense I mean it) is what you did and it shows only
1-2 bands, the problem is likely (#1) outlined above.
6) If all else fails, cut out one or two of the bands from your gel
and run a mass spec. An expensive way to find out that it is
aggregation. Nevertheless....
Hope that helps.
Raji
On Sep 23, 2008, at 3:51 AM, Meg wrote:
> 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.
>
> <SDS PAGE.JPG><IEF.doc>
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