Need help with fluorochrome labelling of antibodies
zxy5 at po.CWRU.Edu
Sat Nov 5 03:46:26 EST 1994
I need to label some monoclonal and polyclonal antibodies
with FITC or any other fluorochrome for FACScan (single laser
excitation at wavelength 488). I have found two
protocols in " Antibodies: A Laboratory Manual".
In the first protocol antibody is prepared at 2 mg/ml in 0.1M
sodium carbonate (pH 9.0). Fluorescein isothiocyanate is dissolved
in DMSO at 1 mg/ml and added slowly to the protein
solution ( 50 microgram per ml of antibody). The mixture is
incubated in dark for 8 hours at 4 degrees. Then NH4Cl is added to
50 mM , incubated for 2 hours at 4 degrees. Finally xylene
cylanol and glycerol are added to 0.1 % and 5% respectively.
Antibody is separated by gel filtration.
In the second protocol DTAF is dissolved in 1 molar sodium
carbonate (pH 9) at 2.5 mg/ml and used instead of FITC. 25
microgram of DTAF is added per ml of antibody solution and
there is no 8 hour incubation but 10 minute mixing at room
temperature. Other steps are the same as the first protocol.
I would like to learn your experiences with these or other protocols
and your comments. Which protocol works better? Which company's
dye do you use? Are there kits available for this purpose? Which
other fluorochromes are there? Which methods are
you using for proteins other than antibodies? (Ex: How do you FITC
label cytokines to stain cells to analyze by FACS? ).
Thanks in advance.
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