Biomedical Engineering Reference
In-Depth Information
Table 1.10.
The UV quantitation of nucleic acids in the presence of pro-
teins
A
280
/A
260
T
F
A
280
/A
260
T
F
1.75
0
1.118
0.86
0.052
0.671
1.60
0.0030
1.078
0.84
0.056
0.650
1.50
0.0056
1.047
0.82
0.061
0.628
1.40
0.0087
1.011
0.80
0.066
0.605
1.30
0.0126
0.969
0.78
0.071
0.581
1.25
0.0146
0.946
0.76
0.076
0.555
1.20
0.0175
0.921
0.74
0.085
0.528
1.15
0.0205
0.893
0.72
0.093
0.500
1.10
0.0240
0.836
0.70
0.103
0.470
1.05
0.0280
0.831
0.68
0.114
0.438
1.00
0.0330
0.794
0.66
0.128
0.404
0.96
0.0370
0.763
0.64
0.145
0.368
0.92
0.0430
0.728
0.62
0.166
0.330
0.90
0.0460
0.710
0.60
0.192
0.289
0.88
0.0490
0.691
1.3 Quantitative Phosphate Determinations
1.3.1 Determination of Inorganic Phosphate
in Biologic Samples
A1N perchloric acid
Solutions/Reagents
B5mM sodium molybdate (Na
2
MoO
4
·
2H
2
O, M
r
241.95)
Important!
Do not use ammonium molybdate!
C isopropylacetate
Standard
10 mM KH
2
PO
4
in 0.5 M perchloric acid
Put 1.5 ml of Soln. B and 2.0 ml of Soln. C into phosphate-free test
tubes. The sample, which should contain not more than 100 nmoles
phosphate, is mixed with an equal volume of Soln. A. Give 0.5 ml of
this mixture to the above mixture of B and C. Shake the obtained
mixture vigorously for 30 s andthenspininacentrifugeforashort
period to separate the phases. To avoid the decomposition of labile
organic phosphates, the extraction should be done at 0
◦
C or below.
The molybdatophosphate complex remains in the organic
phase, which is removed and read at 725 nm against a blank.
Standards are made in the range of 5 - 100 nmoles phosphate
per 0.5 ml.
References
Wahler BE, Wollenberger A (1958) Biochem Z 329:508
