The method by which the fluorescent FITC marks antibodies.

When FITC reacts with the
antibody
protein in an alkaline solution, it is mainly the
protein
that binds the rhamide of lysine to the thiocarbide bond of fluorescent to form the FITC-protein binding, i.e. fluorescent antibody or fluorescent binding. One IgG molecule has 86 lysine residues, generally can bind up to 15 to 20, an IgG molecule can bind 2 to 8 molecules of FITC, its reaction is as follows
FITC-N-C-S-S-N-H2-protein → the FITC-NS-C-N-H2-Protein
commonly used Marshall (1958) method to label fluorescent antibodies, can also be used according to the conditions of Chadwick iso-labeling method or Clark and other (1963) dialysis marking method.
1. Marsshall method
(1) material antibody globulin solution, 0. 5mol/L (pH9. 0) Carbonate buffer, sterile physiological saline, isothion cyanate fluorescent, 1% thiomersal aqueous solution, 50 ml small
beaggle
, 4 degrees C refrigerator, electromagnetic mixer, dialysis bag, glass rod, pH7. 2 or 3. 0 of 0. 01mol/LPBS, etc.
(2) method and step
(1) antibody preparation take the appropriate amount of known concentration of globulin solution in the beech, plus human physiological saline and carbonate buffer, so that the last
immunoglobulin
concentration of 20 mg/ml, carbonate buffer capacity of 1/10 of the total, mixed well, will be burned on the electromagnetic mixer (the speed is appropriate to not be suitable for foam) 5 to 10min.
(2) fluorescent, according to the total amount of protein to be labeled, per milligram of immunoglobulin plus 0. 01mg fluorescent pigment,
analysis
balance to accurately name the required isothione fluorin powder. The following formula can also be used to calculate the amount of immunoglobulin, luciferin, but also to calculate the amount of buffer required.
a. Protein solution: content Amg/m1;
b. Total Protein Quantity (AXB) - Crag.
c. C/20 to C/10 s Dmg (e.g. protein content is less than 20 mg/ml, with C/10; if higher than 20 mg/ml, with C/20).
d. Amount of luciferin FITC: (1/50 to 2/100) XC-Emg.
e. 0. 5mol/L (pH9. 5) Carbonate buffer D/10-Fml.
f. PBS quantity D-(B-F) - Gml.
Note: A is the protein content, mg/ml; B is the volume of the protein solution; C is the total amount of protein, mg; D is constant, mg; is the amount of luciferin, mg; F is the volume of the carbonate buffer, ml; G is the volume of PBS, ml.
(3) combine (or mark) the fluorescent pigment gradually added to the globulin solution, avoid sticking the fluorescent to the wall of the bottle (about 5-10min to finish), after adding, continue to avoid light stirring about 12h. During binding, the protein solution should be kept at about 4 degrees C, so the beech and blender should be moved together into the 4 degrees C refrigerator.
(4) Dialysis After binding, centrifuge the labeled globulin solution (2500r/min) 20rain, remove a small amount of sediment, load it into a dialysis bag, and place it in a berries, using pH8. 0 buffer salt water dialysis (0 to 4 to C) overnight.
(5) cross-pillar to take dialysis overnight markers, through the glucosin gel SephadexG-25 or G-50 columns, separation of free luciferin, collection of labeled fluorescent antibodies for identification. Semployment: 0. 01mol/L Phosphate buffer (pH7. 2);
filtration
: 12 ml labeled global protein solution (not dialysis before filtration); collection: 20 ml (dilution 1. about 7 times).
2. Chadwick
(1)
reagents
and material antibody globulin solution, isothione fluorin, 3% sodium carbonate water solution, 0. 01mol/L pH8.0PBS, 1% thiomersal,
centrifuges
and centrifuges, beech (25ml) blenders, sterile straws, sterile straws and capillary tubes, beasel (500ml) dialysis bags, etc.
(2) method and steps
(1) antibody preparation with o to 4 to C pH8. 0 Phosphate buffer salt water dilutes the globulin solution to a concentration of 30 to 40 mg/ml, placed in a 25 ml beo bowl and placed in an ice tank.
(2) fluorescent pigment preparation per milligram of immunoglobulin added luciferin 0. 01rug calculation, called take the required fluorescent, with 3% sodium carbonate water solution dissolved.
(3) the prepared antibody and fluorescent pigment solution is equally mixed, fully stirred, combined in the o-4 to C refrigerator (preferably in the magnetic mixer continued stirring) 18 to 24h.
(4) dialysis and columnal analysis methods are the same as the Marshall method.
.