2nd posting, affinity chromat. for bFGF

Joy Richman jrichmn at ccu.umanitoba.ca
Sun May 2 16:23:16 EST 1993


This is the second time I am posting this question since I am still
looking for answers to it.

We are attempting to characterize expression of bFGF in the face using
Western blots. 
We are homogenizing our early embryonic chick heads in 0.15 M Ammonium sulfate
with protease inhibitors using a hand held homogenizer, spinning out the
insoluble fraction in a microfuge, and then binding to heparin sepharose
beads under high salt conditions (.6 M NaCl). We are using a batch method
to bind to the beads rather than a column. The final elution is merely to
boil the beads in sample buffer (SDS,glycerol, beta mercaptoethanol and
dyes). Thus we should elute all forms of bFGF including any higher
molecular weight forms. We have had intermittent success with this method.
By success I mean that we can see a 18 kD band in some of our extracts but
not in others. There is alot of variation between preparations. Some
preparations have no low molecular weight bands but only 3-4 very large
molecular weight bands (40-60 kD or greater!) Unfortunately we only have
enough from each preparation to load in one lane of one gel. I am
dissapointed in the lack of reproducibility and wonder if anyone has had
experience with affinity chromatography and can offer suggestions?

Oh our polyclonal antibodies are raised to the first 24 aminoacids of 146
aa form of bFGF conjugated to Keyhole Limpet Hemocyanin.  The antibody
cross reacts with chick and bovine bFGF and works well in our detection
system (125I). We can pick up as little as 1 ng of bFGF. 

Joy Richman


-- 
The most important event in life is not birth or death but gastrulation.
Lewis Wolpert
jrichmn at ccu.umanitoba.ca



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