Biomedical Engineering Reference
In-Depth Information
1.1.5 BCA Protein Determination
This method should be preferred if protein concentration has to
be determined in the presence of detergents. But if copper chela-
tors,suchasEDTA,orreductants,suchas2-mercaproethanolor
DTE/DTT or reducing carbohydrates (e.g.
>
10 mM glucose), are
components of the sample, the test does not work reliably.
1.1.5.1 BCA Standard Procedure
A
1% (w/v) BCA (2,2
-biquinoline-4,4
-dicarboxylic acid, bicin-
Solutions/Reagents
H
2
O, 0.16%
(w/v) disodium tartrate, 0.4% (w/v) NaOH, 0.95% (w/v)
NaHCO
3
, correct pH to 11.25 if necessary with NaOH or
NaHCO
3
.StableatRT.
B4%CuSO
4
·
choninic acid, disodium salt), 2% (w/v) Na
2
CO
3
·
5H
2
O.StableatRT.
C mix 100 vol. of Soln. A with 2 vol. of Soln. B. Stable at least for
1 week at RT.
Standard
:1.0mg
/
ml BSA
4
The standard curve is made between 0 and 100
µ
g.
µ
l with ddH
2
O, and then
add 2.0 ml of Soln. C and incubate the mixture at 37
◦
C for 30 min.
Measuring wavelength is 562 nm.
Fill samples and standards up to 100
1.1.5.2 BCA Micromethod
A
8% (w/v) Na
2
CO
3
·
H
2
O, 1.6% (w/v) NaOH, 1.6% (w/v) dis-
Solutions/Reagents
odium tartrate, correct pH to 11.25 with NaHCO
3
.Stableat
RT
B 4% (w/v) bicinchoninic acid, disodium salt (BCA). Stable at
RT
C4%CuSO
4
·
5H
2
O.StableatRT
Dmix 0vol.ofSoln.Awith48vol. of Soln. B and 2 vol.ofSoln.C
The standard curve is made between 0 and 100
µ
µ
g BSA/100
l.
µ
µ
Fill up 100
l of sample or standard to 100
l with ddH
2
O, if
µ
necessary, and mix with 100
l of Soln. D. Read the absorption after
incubation for 30 min at 60
◦
C at 562 nm.
The incubation time may vary dependent on incubation tem-
perature: 37
◦
C -2h.
This microassay may be done on microtiter plates.
References
Fountoulakis M, Juranvill JF, Manneberg M (1992) J Biochem Biophys Meth
24:265
4
Like by other quantitative methods pure proteins give different results
when the same weight was used, e.g., ovalbumin 93%, rabbit IgG 90%,
mouse IgG 80%, human IgG 97%, and chymotrypsinogen 100% when
compared with BSA (data from Pierce Protein Assay Technical Hand-
book 1996).
