Biomedical Engineering Reference
In-Depth Information
A 0mM CuSO
4
,40mM citric acid, 0.1 mM EDTA
Solutions/Reagents
B .4M Na
2
CO
3
,0.32M NaOH
Cmix1vol. freshly prepared A with 25 vol. freshly prepared B
D Folin-Ciacalteu's phenol reagent (stock), 1 + 1 diluted with
ddH
2
O
E 0
µ
/
ml malachite green in 0.1 M sodium maleate buffer, pH
6.0, 1 mM EDTA
Measure at 690 nm immediately after addition of solution E.
The assay may be done in a microtest plate (Table 1.3).
g
Micro assay
Table 1.3.
L
OWRY
microassay
Blank
Standard
Sample
µ
(
l)
Buffer
Max. 15
-
-
Standard
-
Max. 15
-
Sample
-
-
Max. 15
H
2
O
to15
to15
to15
Solution C
15
15 15
Mix, incubate for 15 min at RT
Solution D
3
3 3
Mix, incubate for 30 - 45 min at RT
Solution E
180
180
180
Detergents, e.g., SDS, at elevated concentrations strongly dis-
turb the test. If at high blank level the difference between blank
and sample is too small, this interference should be omitted by an
extraction of the detergent (cf. Protocol 1.1.2 and 1.1.4).
Prior to the addition of Soln. E, extract the sample twice with
1 ml ethyl ether each. Remove the ether by aspiration after cen-
trifugation; remove remaining ether in the aqueous phase with
a SpeedVac. Prepare the standard curve in the range between 0 and
1
µ
g BSA. This extraction of detergents is not allowed to be done in
a microtest plate.
References
Lowry OH, Rosebrough NJ, Farr AL, Randall RL (1951) J Biol Chem 193:265
Sargent MG (1987) Anal Biochem 163:476
1.1.1.3 Micromethod on Microtest Plates
Between 0.5 and 80
µ
µ
/
ml)
may be estimated in a microtest plate (96-well plate, flat bottom).
A 0g Na
2
CO
3
(anhydrous) in 1000 ml 0.1 N NaOH
g of protein (equivalent to 20 - 1600
g
Solutions/Reagents
B .0gCuSO
4
·
5H
2
O in 100 ml ddH
2
O
