296
M. A. GERENCSER
1. Fractionation of rabbit antiserum (adapted from Hebert et al., 1972, and
Cherry, 1974)
The following procedure is for the fractionation of 10.0 ml of antiserum:
(a) Add 10.0 ml 70% saturated (NH4)2S04 to the serum while stirring
(b) Mix well and allow to stand at 4°C overnight or at 25 "C for 4 h.
(c) Centrifuge and discard supernatant.
(d) Resuspend the globulin precipitate in distilled water to a total
volume of 10.0 ml.
(e) Add 10-0 ml 70% saturated (NH&S04; mix, centrifuge and discard supernatant.
(f) Resuspend globulin precipitate to a total volume of 10.0 ml in
distilled water and repeat step (e).
(g) Resuspend the globulin to a total volume of 10.0 ml or less if
concentration is desired in distilled water.
(h) Dialyse against frequent changes of 0.9% NaCl, pH 8-0 to remove
the (NH&S04. The presence of sulphate can be detected by adding a
small amount of saturated BaClz to an equal volume of dialysate. There
should be no cloudiness in an aliquot of an overnight dialysate. Excess
(NH4)2S04 may also be removed by passing the globulin through a
Sephadex G-25 column.
gently.
2. Conjugation with fluorescein isothiocyanate (FITC) (adapted from
Hebert et al., 1972, and Cherry, 1974)
(a) Determine the protein concentration of the globulin. The biuret
method is satisfactory for this purpose.
(b) Determine the total protein content of the globulin and calculate the
amount of FITC needed for the desired F/P ratio. T o achieve F/P ratios in
the range of 10-12 requires 15 ,ug/mg FITC while ratios of 15-20 require
25 ,ug/mg FITC. These amounts apply when the globulin has a protein
concentration between 10 and 30 mg/ml.
(c) For direct labelling of globulin, bring all reagents to 25 "C.
(d) Dissolve the FITC in buffer (pH 9.0, 0.1 M NazHPOd), using
one-half the volume of the globulin to be labelled; for 10 ml globulin,
dissolve FITC in 5 -0 ml.
(e) Place the globulin in a flask and add 0.2 M Na2HP04 in a volume,
equal to 1/4 that of the original; stir gently; for 10 ml globulin, use 2.5 ml
Na2HP04.
(f) Add the FITC solution while stirring gently.
(g), Immediately adjust to pH 9.5 by dropwise addition of 0.1 M
Na3P04.
M. A. GERENCSER
1. Fractionation of rabbit antiserum (adapted from Hebert et al., 1972, and
Cherry, 1974)
The following procedure is for the fractionation of 10.0 ml of antiserum:
(a) Add 10.0 ml 70% saturated (NH4)2S04 to the serum while stirring
(b) Mix well and allow to stand at 4°C overnight or at 25 "C for 4 h.
(c) Centrifuge and discard supernatant.
(d) Resuspend the globulin precipitate in distilled water to a total
volume of 10.0 ml.
(e) Add 10-0 ml 70% saturated (NH&S04; mix, centrifuge and discard supernatant.
(f) Resuspend globulin precipitate to a total volume of 10.0 ml in
distilled water and repeat step (e).
(g) Resuspend the globulin to a total volume of 10.0 ml or less if
concentration is desired in distilled water.
(h) Dialyse against frequent changes of 0.9% NaCl, pH 8-0 to remove
the (NH&S04. The presence of sulphate can be detected by adding a
small amount of saturated BaClz to an equal volume of dialysate. There
should be no cloudiness in an aliquot of an overnight dialysate. Excess
(NH4)2S04 may also be removed by passing the globulin through a
Sephadex G-25 column.
gently.
2. Conjugation with fluorescein isothiocyanate (FITC) (adapted from
Hebert et al., 1972, and Cherry, 1974)
(a) Determine the protein concentration of the globulin. The biuret
method is satisfactory for this purpose.
(b) Determine the total protein content of the globulin and calculate the
amount of FITC needed for the desired F/P ratio. T o achieve F/P ratios in
the range of 10-12 requires 15 ,ug/mg FITC while ratios of 15-20 require
25 ,ug/mg FITC. These amounts apply when the globulin has a protein
concentration between 10 and 30 mg/ml.
(c) For direct labelling of globulin, bring all reagents to 25 "C.
(d) Dissolve the FITC in buffer (pH 9.0, 0.1 M NazHPOd), using
one-half the volume of the globulin to be labelled; for 10 ml globulin,
dissolve FITC in 5 -0 ml.
(e) Place the globulin in a flask and add 0.2 M Na2HP04 in a volume,
equal to 1/4 that of the original; stir gently; for 10 ml globulin, use 2.5 ml
Na2HP04.
(f) Add the FITC solution while stirring gently.
(g), Immediately adjust to pH 9.5 by dropwise addition of 0.1 M
Na3P04.