 |
 |
 |
 |
Greetings from the U. of Vt. I've just received a call from an investigator
who is going to label platelets with a FITC conjugated marker and an
activation marker linked to Cy5. They are using a kit from Amersham LIFE
SCIENCE called FluoroLink-Ab Cy5 Labeling Kit.The detection of plts. is no
problem, but I've never worked with Cy5. Does anyone have experience with
this kit and/or running such specimens on the flow.
{No experience with the kit, but lots with Cy5. Although you haven't
specified how you're going to be using the reagents, I can provide some
generalities about Cy5. If it's a direct conjugate of the antibody, you need
to excite Cy5 with red light--either a HeNe laser (633nm) or a Kr laser
(647nm) or a diode laser emitting somewhere in this range. Cy5 fluorescence
is best viewed around 670nm; I believe that I had Omega make a 670BP20 for
about $150 some 5 years ago. FITC and Cy5 are a good pair with respect to
fluorescence because there is little appreciable emission overlap of the two
dyes.
{This means that you need a dual laser system or a mixed gas Ar/Kr laser
emitting 488nm and 647nm in order to view FITC and Cy5 associated with the
same cell.
{If you're making a direct conjugate of your own antibody with activated Cy5,
the conjugation is very easy to do. As I recall, separation of free dye from
dye/protein conjugate was very efficient over a G10 column; you may or may
not have to concentrate by dialysis. The conjugation was good in preserving
antibody activity. A dye/protein ratio of around 2 should avoid quenching
problems. Since excess dye will be washed away there will be no residual dye
to stick to tubing etc.
Dave Coder
dcoder@u.washington.edu
|
|
|
|