Biomedical Engineering Reference
In-Depth Information
Table 4.7.
Ammonium sulfate fractionation of sera of different species
Species
Ammonium sulfate saturation (%)
Content
1st Precipi-
2nd Precipi- 3rd Precipi-
tation
a
tation
a
tation
of Ig (%)
Cat
35
35
30
71
Chicken
35
35
35
73
Goat
45
30
-
83
Guinea pig
40
40
35
74
Hamster
35
35
35
68
Horse
30
30
30
45
Man
50
45
45
Mouse
40
40
35
75
Pig
35
35
35
72
Rabbit
35
35
35
91
Sheep
35
35
35
84
a
The amount of ammonium sulfate resulting from the previous precipi-
tation has to be taken into consideration
Data from Jones GL, Hebert GA, Cherry WB (1978) Fluorescent anti-
body techniques and bacterial applications. H.E.W. Publ., Atlanta
media. Note that loss of immunoglobulin subclasses may occur
caused by different binding affinities to the Fc receptors Proteins A
and G, and to the light chain receptor Protein L (cf. Table 4.8).
Antibody complexes are precipitated by relatively low concen-
trations of polyetyleneglycol 6000 (PEG 6000).
Dilute serum with Soln. D in a ratio of 1:25 and add the same
PEG precipitation
volume of Soln. E to the dilution. Incubate at 4
◦
C overnight and
spin with 20 000
g for 20 min. Wash the pellet once with Soln. F
anddissolvetheprecipitateafterafurthercentrifugationinPBS.
×
References
Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring
Harbor Laboratory, New York, p 298
Digeon M, Laver M, Riza J, Bach JF (1977) J Immunol Methods 16:165
4.4 Removal of Unspecific Immunoreactivities
In Western blots and in immunofluorescence, antisera often give
unspecific reactions, especially when rabbit sera are used. These
unspecifitiesmaybesuppressedbypretreatmentwithliverpowder.
Add about 50 mg ofliverpowderto1ml of antiserum and shake
at RT for 15 min. Then spin at 10 000
×
g. The supernatant is ready
for preparing working dilutions.
