Sunday, January 31, 2010

Quantitative Determinations of Proteins

                                                                        بسم الله الرحمن الرحيم


ovalbumin (Ova) or bovine serum albumin (BSA) in 0.1% SDS
(w/v) is suitable. This solution can be stored in the refrigerator for
several weeks.
A 20g Na2CO3 (anhydrous) in 1000 ml 0.1 N NaOH Solutions/Reagents
B 1.0g CuSO4 · 5H2O in 100 ml ddH2O
C 2.0g potassium-sodium tartrate in 100 ml ddH2O
D mix1vol. B and 1 vol. C, and then add 50 vol. A
E Folin–Ciocalteu’s phenol reagent (stock), 1+1 diluted with
ddH2O
Standard 5.0 mg/ml ovalbumin or BSA, 0.1% SDS (w/v) in ddH2O
Table 1.2. LOWRY standard protocol
Blank Standard Sample
(ml)
– Max. 0.1 –
– – Max. 0.1
H2O 0.1 to 0.1 to 0.1
Soln. D 2.0 2.0 2.0
Mix, incubate for 5–10 min at RT
Soln. E 0.2 0.2 0.2
Mix, incubate for 30–45 min at RT, read at 700 nm
Make samples, blank, and standards at least in duplicates, and
Standard protocol
measure in a spectrophotometer at 700–750 nm.
Especially for small amounts of protein, reduce the volumes: Half-micro protocol
0.1 ml of 0.1% SDS in ddH2O are added to 0.10 ml of sample, and
then add 1.0 ml Soln. E, and 5 min thereafter add 0.1 ml Soln. D.
Measure after 30–45 min.
Prepare the standard curve in the range between 0 and 30 μg
of protein. Since the standard curve in this range is nearly linear,
it is possible to take a factor F, which can be estimated at that time
when solution D is used for the first time.
μg Protein/100 μl = ASample − ABlank · F
Mix suspensions of membrane proteins, cell homogenates, etc.,
with an equal volume of 0.1 NaOH to get a homogenous solution.
For estimation of proteins covalently bound to chromatographicmatrices
hydrolyze the sample for 6 h at 37 ◦Cin solutionD.
After centrifugation, use an aliquot for protein determination.
1.1.1.2 Modification by SARGENT
A50-fold increase in sensitivitywith respect to the Lowry standard
protocol was described by Sargent. It is possible to estimate 0.1–
1 μg protein and 4–40 μg/ml, respectively.
Solutions/Reagents A 20mM CuSO4, 40mM citric acid, 0.1mM EDTA
B 0.4MNa2CO3, 0.32MNaOH
C mix1vol. freshly prepared A with 25 vol. freshly prepared B
D Folin–Ciacalteu’s phenol reagent (stock), 1+1 diluted with
ddH2O
E 60μg/ml malachite green in 0.1Msodium maleate buffer, pH
6.0, 1mM EDTA
Measure at 690 nm immediately after addition of solution E.
The assay may be done in a microtest plate (Table 1.3).
Table 1.3. LOWRY microassay
Blank Standard Sample
(μl)
Buffer Max. 15 – –
Standard – Max. 15 –
Sample – – Max. 15
H2O to 15 to 15 to 15
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-
Micro assay
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 centrifugation;
remove remaining ether in the aqueous phase with
a SpeedVac. Prepare the standard curve in the range between 0 and
1 μg BSA. This extraction of detergents is not allowed to be done in
a microtest plate.
References
LowryOH, RosebroughNJ, Farr AL, Randall RL (1951) J Biol Chem 193:265
Sargent MG (1987) Anal Biochem 163:476
1.1.1.3 Micromethod onMicrotest Plates
Between 0.5 and 80 μg of protein (equivalent to 20–1600 μg/ml)
may be estimated in a microtest plate (96-well plate, flat bottom).
Solutions/Reagents A 20g Na2CO3 (anhydrous) in 1000 ml 0.1 N NaOH
B 1.0g CuSO4 · 5H2O in 100 ml ddH2O

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