Re: "sticky collagen"

<< Previous Message | Next Message >> (Peter van de Plas) (by way of histonet)
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Dear Patsy,

Sorry you are wrong, I am not "one of you Brit's" :)
Please find included a copy of the answer I sent to the histonet after a
question of Anita Jennings last year:

Here it comes:
"The different blocking steps you have tested all block hydrophobic areas
("sticky sites") in your specimen. Hydrophobic areas are blocked before the
immunoincubation with e.g. normal serum and BSA. Once blocked these sites
generally will not give rise to background anymore.

Cartilage and perichondrium are composed of collagen fibers with a strong
positive charge (still present after aldehyde fixation) embedded in
proteoglycans which have a strong negative charge. Most antibodies
(primaries and secondaries) are negatively charged at pH 7-8.2. I therfore
think that the collagen fibers present in the cartilage tissue are causing
your background problem. This so-called charge determined background can be
circumvented by adding negatively charged molecules (e.g. aurion BSA-c) to
wash and incubation buffer. The other possible cause for background
(aspecific binding to proteoglycans) can be prevented by adding gelatin to
your buffers. (do not put both BSA-c and gelatin in the same buffer since
they will have charge determined affinity to each other as well)

I invite you to visit our web-site for detailed info on  the topic above.
When I can be of further assistance please let me know."

It turned out (when I recall it correctly) that in Anita's case the
dilution of the primary antibody was causing the background problems.

Hope this is of help
Greetings from the Netherlands, Peter

Peter van de Plas
Costerweg 5
6702 AA Wageningen
The Netherlands
phone: (31)-317-497676
fax: (31)-317-415955

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