Biomedical Engineering Reference
In-Depth Information
Add 10 ml glacial acetic acid to the washed pellet and boil to re-
flux for 2 h.CooltoRTanddeterminethevolume.Readabsorbance
at 340 nm.
Molar absorption coefficient of the formed sulfonamide
ε
=
340
ρ
=
/
10
4
M
−1
cm
−1
; specific gravity of wet Sepharose
1.24
ml.
Bound amino spacer or immobilized protein may be deter-
mined also by the Kjeldahl method (Protocol 1.1.6).
×
1.1 g
References
Antoni G, Presentini R, Neri D (1983) Anal Biochem 129:60
3.6.2.2 Quantitative Determination of Immobilized Protein
The BCA assay for immobilized protein is more reliable than cal-
culations from UV measurements of coupling supernatant.
A
141 mM Na
2
CO
3
, 113 mM NaHCO
3
, 100 mM NaOH, 7 mM
Solutions/Reagents
sodium tartrate, pH 11.25
B 1% BCA (w/v), disodium salt, in ddH
2
O
C4%
5H
2
O (w/v) in ddH
2
O
D 5vol. Soln. A, 24 vol.Soln.B,and1vol. Soln. C; mix prior to
use
Standard
1 mg
SO
4
·
/
ml BSA in Soln. A
Wash the affinity matrix after coupling and blocking as described
in Protocol 3.6.2. In a second step, wash about 50
µ
l of the support
with 1 ml of Soln. A. Remove excess solvent by spotting onto filter
paper (avoid drying).
Weigh triplicates of 5 mg of wet support into 1.5-ml reaction
tubes, add 500
µ
µ
l of Soln. D
2
.Shake
at 60
◦
C for 1 h, and then read absorbance at 562 nm against water.
Thestandardcurveismadefrom0to50
l of Soln. A followed by 500
µ
g BSA in Soln. A.
References
Kakabakos SE, Tyllianakis PE, Evangelatos GP, Ithakissios DS (1994) Bio-
materials 15:289
3.6.2.3 Immobilization of Wheat Germ Agglutinin
A .1M N-acetyl-glucosamine (GlcNAc, M
r
221.2), 1 M NaCl,
Solutions/Reagents
0.1 M borate buffer, pH 8.3
B .5Methanolamine (3 ml 2-aminoethanol in 80 ml pure water,
adjust pH to 8.0 with HCl, bring up to 100 ml with pure water)
C .5M NaCl in 0.1 M carbonate buffer, pH 9.0
D .5M NaCl in 0.1 M acetate buffer, pH 4.5
E
0.9% NaCl (w/v), 0.02% sodium ethylmercurithiosalicylate
(Thimerosal) in ddH
2
O
2
The same volumes are used when commercially available BCA reagents
are used.
