Protein determination according to Lowry procedures for protein solution with interference, or for membrane extracts.
Peterson has described a precipitation step that allows the separation of the protein sample from interfering substances and also consequently concentrates the protein sample, allowing the determination of proteins by Lowry in dilute solution. Peterson, G. L. (1983) Determination of total protein. Methods Enzymol. 91, 95-121
Interfering Reagents for Lowry: many detergents, Urea, Guanidine HCl, high sucrose, Ammonium sulphate, >0.1M TrisHCl, >1MNa Acetate or Na Phosphate, EDTA, reducing agents, etc.
A) 0.4% CuSO4.5H20 (Keep at 4°C)
B) 0.8% K-Tartrate (Keep at 4°C)
C) 20% Na2CO3 (Keep at RT)
D) 10% SDS (Keep at RT)
E) 0.8 M NaOH (Keep at 4°C)
F) Folin Ciocalteus (Keep at RT)
G) 15% DOC (deoxycholate). Prepare fresh
H) 72% TCA (Keep at 4°C)
I) 1 mgr BSA (Bovine Serum Albumin) per ml H20 (Keep at -20°C)
1) Complex-forming reagent
Prepare immediately before use by mixing the following stock solutions:
0.25 Vol A + 0.25 Vol B
Add slowly 0.5 Vol of C with agitation
Add 1 Vol of D
Add 1 Vol of E
Add 5 Vol of H20
2) Dilute Folin
Dilute Folin reagent (F) 1:6 in H20. Prepare immediately before use.
1) Sample: Load between 2 to 8 µgr of the protein sample in 1.5ml plastic tubes. Complete with H20 up to 0.5 ml
2) Blank and Standarts: Load in 1.5ml plastic tubes:
0 µgr BSA: blank
2 µgr BSA: 2 µl BSA 1 mgr/ml
4 µgr BSA: 4 µl BSA 1 mgr/ml
5 µgr BSA: 5 µl BSA 1 mgr/ml
6 µgr BSA: 6 µl BSA 1 mgr/ml
8 µgr BSA: 8 µl BSA 1 mgr/ml
Complete with H20 up to 0.5 ml
3) Add 50 µl DOC 0.15% (G). Mix and keep 10 min RT.
4) Add 50 µl TCA 72% (H). Mix. Keep 10 min RT.
5) Spin 25 min at approx.13000 rpm.
6) Throw supernatant and dry pellet by inversion (on paper tissue).
7) Resuspend pellet in 400 µl complex-forming reagent using a vortex mixer, and let the solution stand at room temperature for 10 min
8) Add 0.1ml of diluted (1:6) Folin reagent, using a vortex mixer, and let the mixture stand at room temperature for 30-60 min
9) Read at 750 nm. Plot a standard curve of absorbance as a function of initial protein concentration and use it to determine the unknown protein concentrations.
1. The incubation period is not critical and can vary from 10 min to several hours without affecting the final absorbance.
2. The Vortex step is critical for obtaining reproducible results. The Folin reagent is only reactive for a short time under these alkaline conditions, being unstable in alkali, and great care should therefore be taken to ensure thorough mixing.
3. The assay is not linear at higher concentrations. Ensure, therefore, that you are analyzing your sample on the linear portion of the calibration curve.
4. A set of standards is needed with each group of assays, preferably in duplicate. Duplicate or triplicate unknowns are recommended.
5. The amount of color produced in this assay by any given protein (or mixture of proteins) is dependent on the amino acid composition of the protein(s) (see Introduction to Lowry Method). Therefore, two different proteins, each for example at concentrations of 1 mg/mL, can give different color yields in this assay. It must be appreciated, therefore, that using BSA (or any other protein for that matter) as a standard only gives an approximate measure of the protein concentration. The only time when this method gives an absolute value for protein concentration is when the protein being analyzed is also used to construct the standard curve. The most accurate way to determine the concentration of any protein solution is amino acid analysis.
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