There were 2 or 3 main solutions to the question regarding high backgrounds
for PE-Cy5 conjugates. There was one answer sent to the list, I believe,
that offered an alternate method of searching the Purdue archives using
http://www.alltheweb.com , I share this as this was included in my question.
1) Several companies offer PE-Cy5 conjugates which have less background
than earlier PE-Cy5 conjugates. Caltag says their PE-Cy5 conjugates have
been reformulated to solve this problem, as well as Beckman Coulter (from
the original AMAC company), and Sigma which have a blocking component to
the Fc binding of the dye.
2) A few people offered the idea of blocking with Cy5, BSA-Cy5 provided
that a Red LASER line isn't being used. (100ug was the suggested
concentration in one e-mail).
3) A recommendation was also given to dilute the PE-Cy5 conjugates as low
as possible while still obtaining a good signal, and the background should
be able to be titrated out.
4) Some also suggested spinning the antibody at high speed which should
reduce background.
5) Some suggested that the background can be minimized by use of Ig or an
anti-Fc receptor antibody.
Just for information, one e-mail mentioned that the CY-5 binds to the FcR
I, CD64. This may also be useful information.
I hope that this information is helpful to others.
Doug
{-----------------------------------------------------------------------}
Douglas Smoot
NIDDK-Navy Transplantation & Autoimmunity Branch
Naval Medical Research Center
AFRRI Building 46, Room 2415
8901 Wisconsin Avenue
Bethesda, MD 20889-5603
voice: 301-295-1843
fax: 301-295-6484
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