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>
