Biomedical Engineering Reference
In-Depth Information
µ
Add 33
l of fluorescein isothiocyanate (FITC; tetramethylrho-
damine isothiocyanate, TRITC, or another fluorescent dye isothio-
cyanate derivative is used the same way), 50 mg
/
ml in DMF, to 1 ml
/
of 5 mg
ml IgG in Soln. A. Shake at RT protected from light for 1 h.
Remove surplus FITC and its hydrolysis products on a Sephadex
G-25 column, equilibrated with PBS. The conjugate appears in the
void volume and should be concentrated by ultrafiltration. Add
sodium azide to a final concentration of 0.02% (w/v) and glycerol
up to 10% (w/v) and store at 4
◦
C.
A selection of fluorescent dyes is given in Table 4.3.
Calculate the degree of substitution F/P (nmoles FITC per
nmoles IgG) using the following equation:
A
495
·
V
·
13.1515
·
160
=
F/P
P
A
495
, absorption of the sample at 495 nm and 1 cm path; V, total
volume of the conjugate in milliliters; P, total amount of IgG in
micrograms.
AgoodconjugatehasanA
495
/A
280
quotient of 0.3 - 1.0.
The protein concentration P is determined by measuring the
sample dissolved in PBS (reference: PBS) at 280 nm with consider-
ation to the fluorochrome-specific absorption at 495 nm:
A
280
·
(0.35
·
A
495
)
·
dilution
=
/
Fluorescein:
P
(mg
ml)
1.38
A
280
·
(0.56
·
A
550
)
·
dilution
=
/
Tetramethylrhodamine: P
(mg
ml)
1.38
The ratio F/P (moles fluorochrome per milligram of protein) is
calculated by
A
495
A
280
− (0.35
2.87
·
=
Fluorescein
F/P
·
A
495
)
A
550
A
280
− (0.56
4.47
·
=
Tetramethylrhodamine
F/P
·
E
550
)
/
P: protein concentration in mg
ml;A
280
,A
495
,A
550
: absorption at
280, 495 (fluorescein), and 550 nm (tetramethylrhodamine), re-
spectively; 1-cm path, blank PBS; dilution: dilution of the sample
(dilution is 101, if 10
µ
µ
l of conjugate solution are added to 1000
l).
References
Friemel H (ed.) (1991) Immunologische Arbeitsmethoden. Gustav Fischer,
Jena, p 530
Hermanson GT (1996) Bioconjugate Techniques. Academic Press, San
Diego, p 303
Hudson L, Hay FC (1989) Practical Immunology. 3rd ed. Blackwell Sci.
Publ., Oxford, p 34
