FITC label procedure for Toxoid???

[Kim Sabelko]

pemanuel at umabnet.ab.edu (Peter) wrote:

>  I have a toxoid proetin which I must label with FITC.  I am seeking a
>procedure which I can use to purify the unbound away from the labelled
>stuff.  I have been given a procedure which involves a lot of columns.  It
>is also dated 1976.  I cannot believe that no one has developed a better
>way than running a set of columns.
>  Does anyone know of a good procedure paper on FITC conjugation?
>Any references or advice from experienced FITC users would be
>appreciated.  No comment is too trivial for me.  Well almost no comment. 
>Anyway let me know if there is any easier way I can look into.


To purify the FITC conjugated material you might want to try dialysis.
Current Protocols in Immunology recommends dialyzing in 500 ml dialysis
buffer (0.1M Tris.Cl, pH7.4, 0.1% (v/v) NaN3, 0.2 M NaCl; pH adjusted to 7.4
with NaOH) @ 4 C x 48 hrs, with 2-3 changes of buffer.  You can easily
monitor the dialysis by the change in color of the buffer -- the first 1-2
changes it will be yellow.  

Reference:  Current Protocols in Immunology, Chp. 5 Immunofluorescence and
Cell Sorting, pp 5.3.2-5.3.3

Good luck-- Kim