Enzyme pretreatment, and all steps in epitope retrieval, should follow the same
quality control steps as antibody incubation and antibody concentration.
Enzyme pretreatment is not magic, however the kinetics involved are very
difficult to predict. Epitopes are typically chemical shapes within tertiary
protein structure, however they can involve secondary structure and quartinary
structure. The purpose of enzyme pretreatment is to get the right epitope in
the right configuration so it can be recognized by the antibody. Heat
retrieval is meant to break formalin cross linkages, but enzyme pretreatment
actually eats away at part of the protein (depending on the protease).
Too little protease treatment and it does nothing, too much and the epitope is
destroyed.
The bottom line, novel antibodies need to be validated with numerous retrieval
methods. If it is deemed that a protease is better, numerous times and
numerous concentrations should be tried -- even at different temperatures.
Finally, there is no reason to believe that different novel antibodies will
require the same pretreatment, however, a common pretreatment may be
sufficient (though possibly not optimal) for each antibody.
One last thing, if you know more about the nature of the antigen used to create
the antibody, you may be able to predict the required pretreatment -- talk to
the primary investigator.
Will Chappell, HTL(ASCP)QIHC
Anatomic Pathology Supervisor
Children's Hospital of Orange County
On Jul 31, 2012, at 8:41 PM, Young Kwun wrote:
Dear Histonetters,
Could anyone explain about the difference between proteolytic enzymes
for immuostaining?
We use enzyme pretreatment rarely nowadays, and apart from some
ready-to-use one (Dako's Proteinase K), I have used Protease (Sigma)
previously when I did manual staining.
At the moment I am using Leica's BondMax autostainer and their enzyme
pretreatment kit (Trypsin ??, usually one drop per 7 ml vial). I know
the pretreatment condition would be affected by the concentration of
enzymes, pH, temperature, incubation time etc.
My question is that do they have different mode of action on tissues? I
am helping a research project and our antibody includes various clones
of Integrin 6 subunit and uPAR.
I have tried enzyme pretreatment with autostainer and manual staining
with Proteinase K (Dako). It seems that some antibodies work better with
certain enzymes.
I mean that some antibodies work well after BondMax enzyme treatment,
but some antibody works better with proteinase K pretreatment manually.
I am using the same polymer detection system (Leica Microsystem) for
both methods.
I would like to find an enzyme which works for both of our antibodies at
the same time.
Thank you.
Young Kwun
Senior Hospital Scientist
Immunohistochemistry
Dept. of Anatomical Pathology
Concord Repatriation General Hospital
Concord NSW 2139 Australia
02-9767-6075 (Tel)
02-9767-8427 (Fax)
young.k...@sswahs.nsw.gov.au
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