Need help with fluorochrome labelling of antibodies

Zafer Yildirim 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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