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Posted on Monday, May 03, 2004
The Following Protocol can be used to couple either FITC or Biotin to IgG. The same method is used for both, and when discrepancies occur, they will be noted.
1. Dissolve 2mg of IgG in 1mL of PBS (pH 8.0) in clean 16x125mm polypropylene test tube. If needed, adjust pH to 8.0 using 0.5M carbonate buffer.
2. Prepare fresh, 1mg/mL of either NHS-FITC in DMSO or 1mg/mL NHS-LC-Biotin in DH2O. If FITC coupling, add 75uL of FITC/DMSO solution to Antibody while vortexing. If Biotinylating, add 150uL of Biotin/Water solution to Antibody while vortexing.
3. Incubate at room temperature for 45minutes.
4. The next step is to separate the coupled from uncoupled FITC or Biotin. This can be done in 2 ways. The first is to Dialyze the coupled antibody against PBS for 24 hours with 3 changes of PBS. Doing this will not change concentration of original antibody (recommended for antibody with a concentration less than 1mg/mL). The second way is to use a PD-10 column. Using this method will dilute the antibody by a factor of 2.
Using a PD-10 column:
a)Wash PD-10 column with 30mL of PBS.
b) Add the 1mL FITC-Ig or Biotin Ig solution after the top of column is almost dry. Close stopcock to stop flow after the Antibody has eluted into the column.
c) Add 5mL of PBS on top.
d)Open Stopcock. collect in 2mL fractions, discarding the first 2mL and keeping the 2nd 2mL fraction. The second 2mL fraction contains the conjugated antibody*.
*NOTE: Step d) appropriate only if 1mL of conjugated-Ig is added to the column. If 2mL of conjugated-Ig is added, discard first 1mL and keep the next 4mL. The antibody is always diluted by 2x after leaving the column.
5. When FITC coupling an antibody, you can determine the FITC to Protein ratio. When Biotinylating an antibody, you can test the antibody by Flow Cytometry.
6. F/P Ratio:
a) Dilute an aliquot of FITC-Ig complex with buffer so that the Absorbance at 280nm (A280) is less than 2.0
b) Determine and record A280 and A492.
c) Calculate protein concentration: protein (mg/mL) = (A280- (A492 x 0.35))/1.4
d) Calculate moles of protein: = protein(mg/mL)/1.5x10^5
e) Calculate moles of FITC: = A492/0.69x10^5
f) Determine F/P ratio: = moles of FITC/moles of protein
g) F/P ratio should be between 5:1 and 6:1 for Flow Cytometric use.
Submitted by: manny440