Protein Quantification: Estimation Assays, Essay Example

Estimation assays, used to determine protein concentration are commonly used in life science research. Estimation of protein concentration is essential in protein purification, cell biology, electrophoresis, molecular biology, among other research applications. Such assay methods include biuret method, lauret assay, Bradford and turbidity method.The Biuret assay the principle of the biuret assay is that substances with two or more peptide bonds form a purple complex with copper salts in the reagent under alkaline conditions.

The Lowry Protein Assays

The Lowry holds the same principle as biuret assay that copper forms complexes with the protein molecules under alkaline conditions and on addition of folin phenol reagent which is a complex compound of phospho-molybdic-phosphotungstic reagent the Folin has princ-phenol reagent which forms bonds with the protein. The bound reagent is undergoes a slow reduction hence color changes from yellow to blue.  This procedure is a less preferable assay than others since it suffers from interference by a broad range of chemicals like. CAPS, barbital, cesium chloride, cysteine, citrate, diethanolamine, EDTA dithiothreitol, EGTA, mercaptoethanol, HEPES, phenol, Nonidet P-40, polyvinyl pyrrolidone, sodium salicylate, sodium deoxycholate, thimerosol, Tricine, TRIS and Triton X-100.The standard should be similar to the unknown because of variations in the intensity of color development between different proteins.  For example, when measuring serum, bovine serum albumin should be used as standard because albumin forms a significant component of serum.

The Bradford Protein Assays

Bradford procedure is a more accurate assay dependent on the ability of protein to bind Coomassie Blue G-250 dye which exists in two colored forms, blue and red. When bound to protein the red form is converted to the blue form which has an absorption maximum at 595 nm.  The high extinction coefficient of the protein-dye complex gives the assay great sensitivity (approximately 4 times that of the Lowry Method).  The dye-binding method is very rapid with very few biological constituents interfering with the method.

Turbidimetric Method

When a protein is mixed with low concentrations of protein precipitant it produces turbidity which can be used as an index of protein concentration. In this method Samples are mixed with potassium ferrocyanide, dilute trichloroacetic acid, sulfosalicylic acid, glacial acetic acid or other organic acids.   After 10 mins, turbidity reaches maximum and is determined spectroscopically at 600 nm.  Turbidimetric techniques are rapid and convenient but results vary with different protein depending on each protein’s solubility in the organic acids.  In addition, they do not differentiate between protein and other acid-insoluble compounds like RNA and DNA.

Materials

The following materials were used: protein of unknown concentration, Bovine serum albumin (BSA) stock solution (10 mg/mL), test tubes, pipets,  test tube racks, Spectronic 20, Spectronic 20 cuvettes, marker pen ,reagents for each assay .

Reagents for Biuret Assay

The  biuret reagent made of  3 gm Copper sulfate pentahydrate x 5 H2O (f.w. 249.68), 9 gm Sodium potassium tartrate (f.w. 282.22) and  5 gm Potassium iodide (166.0),  were dissolved  all in sequence in 400 ml of a low concentration 0.2 M NaOH (f.w. 40.0) an brought to final volume and black precipitate discarded .

Stock Solutions for Lowry Assay

Lowry B: 1% CuSO₄ in diH₂O ,lowry A: 2% Na₂CO₃ in 0.1 M NaOH and Lowry C: 2% sodium potassium tartarate (NaKC₄H₄O₆• 4H₂O)

The following reagents were used.

Lowry Stock Reagent

• 49 ml Lowry A
• 0.5 ml Lowry B
• 0.5 ml Lowry C

Dilute in the ratio 1:1 in diH₂O before use.

The standard used was Bovine serum albumin (BSA).

Folin’s Reagent: Phenol reagent – 2N (Folin – Ciocalteau reagent) which is diluted 1:1in distilled water.

Procedure

Biuret Assay: 1 mL of sample (or 1 mL H₂O in the case of the blank) was added to each labeled test tube and then 4 mL of Biuret reagent was also added.  Thorough mixing was done and the solution allowed to stand at room temperature for 30 minutes. Determine the absorbance at 560 nm.

Lowry Assay: 1 mL of sample (or 1 mL H₂O in the case of the blank) was added to each labeled test tube and then1 mL of Alkaline Copper reagent was also added and mixing done thoroughly. The solution was then allowed to stand for 10 minutes.  4 mL of “Folin working solution” was added and then incubated at room temperature for 30 minutes. Absorbance was determined at 660 nm.

Turbid metric Assay: 1 mL of sample (or 1 mL H₂O in the case of the blank) was added to each labeled test tube followed by 4 mL of 2.5% SSA reagent. Mixing was done thoroughly and allowed to stand at room temperature for 10 minutes.  Absorbance was determined at 660 nm.

Dye-binding Assay:  1 mL of sample (or 1 mL H₂O in the case of the blank) was added to each labeled test tube followed by 5 mL of Bradford reagent. Mixing was done thoroughly and solution allowed to stand at room temperature for 2 minutes. Absorbance was determined at 595 nm.

Results

Methods (BSA Dilutions)   0.01           0.05          0.1              0.5            1             5             10          unknown

———————————————–.——————————————————————————————-

Biuret Assay                        – 0.015      -0.020      -0.003        0.019    0.014     0.013        0.012           0.008

————————————————-.—————————————————————————————–

Lowry Assay                         -0.40       -0.13        0.146         0.224    0.101     -0.15          0.142             -0.26

——————————————————————————————————————————————

Turbidimetric  Assays          .00          0.008       0.008        0.001      0.005      0.022       0.032             0.049

Dye Binding(Bradford)       0.276    0.476        0.456    0.564    0.566    0.570      0.570       0.424

Work Cited

Gornall, AG, CS Bardawill, and MM David. J. Biol. Chem. 177: 751. 1949.

Layne, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins. Methods in Enzymology 10: 447-455. 1957.

Robinson, HW and CG Hogden. J. Biol. Chem. 135: 707. 1940.

Slater, RJ (ed.). Experiments in Molecular Biology. Clifton, New Jersey: Humana Press, 1986. P. 269.

Weichselbaum, TE. Am. J. Clin. Pathol. Suppl. 10: 40. 1946.