Biomedical Engineering Reference
In-Depth Information
equilibrate at RT for at least 2 days; make sure to have a sedi-
ment of solid ammonium sulfate within the bottle; adjust pH
7.0 with ammonia)
C PBS
D .1M Na
2
B
2
O
7
, 0.02% (w/v) NaN
3
or Thimerosal, pH 8.4
E
10% (w/v) PEG 6000 in D
F
5% (w/v) PEG 6000 in D
Spin two 0.7-ml aliquots of (rabbit) serum at 4
◦
C and 15 000 rpm
for 10 min. Join the supernatants and add 1/10 of volume of Soln. A.
Determine volume. Add dropwise and slowly the same volume of
Soln.Btogivea50%saturation.ShakeatRTfor1h andspinas
described above. Carefully remove the supernatant, discard it, and
resolve the precipitate with 1 ml ddH
2
O. Determine the volume
and add 40% of this volume of Soln. B. Allow to precipitate in the
refrigerator overnight. Collect the precipitate by centrifugation,
dissolve the pellet with ddH
2
O, and dialyze against PBS or TBS.
Whereas antibody solutions are stable at 4
◦
C for a longer period,
aliquot the dialysate, determine immunoglobulin content by UV
reading (equation d), Protocol 1.1.7, (absorption coefficients see
Table 4.6), and store the aliquots at −70
◦
C.
For fractionation with low contamination by serum proteins
of sera from other species the percentage of saturation given in
Table 4.7 should be used.
Alternatively, immunoglobulins may be purified by affinity
chromatography on Protein A, Protein G, Protein L or thiophilic
Table 4.6.
Absorption coefficients of immunoglobulins of different
species at pH 7.4
A
1mg
/
ml
1cm
λ
Species
Type of IgG
(nm)
Chicken (yolk)
IgY
1.35
275
Goat
IgG
1.3
280
IgM
1.3
280
Guinea pig
IgG1
1.357
278
Man
IgG
1.38
280
IgM
1.45
280
Mouse
IgG
1.34
280
Fab
1.4
278
IgA
1.35
275
Rabbit
IgG
1.35
280
Rat
IgG
1.46
280
IgM
1.25
280
Sheep
IgG
1.22
280
Data from Fasman G (ed.) (1992) Practical handbook of
biochemistry and molecular biology. CRC Press, Boca
Raton, Florida, p 265
