XIAOMING YANG wrote:
>> I had purified a human protein from two different cell lines. The way i
> took was to employ two different columns: DEAE sepharose and DNA agarose
> columns. But, the results show that there are other two more bands on
> SDS-PAGE gel. I want to take immunopercipitation way to make a further
> purification. How can I do? Protocal? Thank!
The first thing I would do is to make a western blot to see whether or
not the antibody binds to the other bands (this could happen for example
after partial proteolysis). If they don't, you can use the antibody for
Immunoprecipitation is suitable only for very small scale preparations
(ng-ug amounts). You incubate the protein with the antibody, then fish
out the complex with Protein A (or Protein G) agarose beads. Wash a
couple of times with high salt/mild detergent, then use the pellet
directly for example for SDS-PAGE. Alternatively, you can use dead
Staph. aureus bacteria (Pansorbin, Calbiochem) instead of Protein A
agarose (cheaper). Calbiochem has a free booklet on Pansorbin, which
contains suggestions for washing buffer composition.
For larger scale preps, couple your antibody to activated agarose
(several chemistries are available commercially, for example from
Pharmacia) and pack the gel into a small column. Load the column with
your sample, wash with high salt/mild detergent and elute with a
denaturing buffer (Diiodosalicylate or Glycine/HCl pH 4). In any case,
have some neutralising buffer in the tubes you use to collect the
fractions to minimize exposure of your protein to the elution buffer and
thoroughly wash the column with PBS or similar immediately afterwards.
With luck, both your protein and the antibody will withstand this
procedure without permanent damage.
If you find the different bands again after these procedures, then they
may be part of an multi-subunit protein complex.