Recommendations for light level protein detection?

Kenneth J McNeil mcnei002 at maroon.tc.umn.edu
Thu Sep 8 10:14:55 EST 1994


Sorry if you read my previous post. Somehow the mailer left out most of the
article.

I have been attemptin to localize antigens in plant tissue with antibodies
using silver enhancement of gold particles and, so far, have had no positive
results. Western blots show a strong reaction with a single band (6.5 kD) in
tomato anthers. Primary antibody dilution in Western blots is 1:100, secondary
antibody (antirabbit-HRP) is 1:2,000.
I have tried various dilutions and methods of fixation (paraformaldehyde,
glutaraldehyde), as well as fresh tissue. I have made sections of
paraffin-embedded tissue as well as OCT-embedded tissue for cryostat
sectioning. Section thickness is 10 microM.
Detection of antigens with antibodies using light microscopy is with silver
enhancement of 4 nM gold particles. Primary antibody dilution is 1:20 up to
1:100 and antirabbit-gold (Jackson ImmunoResearch) is 1:50 (manufacturer
recommends 1:20-1:200). Silver enhancement using the method sent by Jackson
ImmunoResearch produces no detectable differences in sections when
recommends 1:20-1:200). Silver enhancement using the method sent by Jackson
ImmunoResearch produces no detectable differences in sections when
compared to the control (no primary antibody). Some silver particles are
seen scattered throughout the control and treated sections, but no specific
regions are detected. The Intense kit from Amersham gave similar results.
Western blots detected with primary antibody (1:100) and antirabbit-4nM gold
(1:200) did show the 6.5 kD band when detected by silver enhancement.
High endogenous peroxidase activity has made use of horseradish peroxidase
difficult. Treatment of sections with hydrogen peroxide (to inactivate
endogenous peroxidase) has not been successful, I still see endogenous
peroxidase. We have considered using fluorescent conjugated antibodies,
but the autofluorescence of some tissues in the anther may be another
problem that I then have to deal with.

Any recommendations or suggestions for what to do next? Is there a better
method for detection of Abs at the light level?

Thanks for your help,
Ken
kmcneil at molbio.cbs.umn.edu



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