Estimation of protein concentration Essay
Estimation of protein concentration
Protein assays are designed to measure the total protein in a solution. Protein assays are quantitative if the protein to be assayed is available in sufficient quantity such that one is able to use it to create a standard curve. If this cannot be achieved, then a standard protein, such as albumin, may be used for a standard curve with the understanding that the results on the unknown protein are semi-quantitative. Since most proteins are not available in large quantities, standard curves for protein assays are typically based on the use of either bovine serum albumin (BSA) or bovine gamma globulin (IgG). The Bradford protein assay is a method to determine protein concentration in the mixture. This method is based on the proportional binding of the dye Coomassie to proteins. If there is a higher protein content, the more Coomassie binds and it produces a significant change in colour of the mixture. The Bradford protein assay contains the dye Coomassie Blue G-250 which is red brown colour in acidic solution. When the protein binds to the dye, the pKa of the dye shifts and causes the dye to turn blue. The maximum absorbance spectrum of the dye is at 595nm. According to Beer-Lambert Law, a standard curve of absorbance versus protein concentration produced by measuring the absorbance of the protein solutions of a known concentration is often used to estimate the protein concentration of an unknown solution. Other than that, the Bradford protein assay reacts variedly with the composition of the protein and is also sensitive to non-protein sources, particularly detergents, and becomes nonlinear with higher protein concentrations.
The two advantages of using Bradford protein assay compared to other methods are, first, Bradford method involves fewer mixing steps and secondly, it does not require heating. However, Bradford method has its disadvantage in measuring protein concentration, that is, dilution is needed before analysing the concentration of the protein. This is due to a linear short range which is typically around 0 µg/ml to 2000 µg/ml. The result of the protein concentration of this experiment which is 51.25 µg/ml and nutritional information of the apple juice which is 2000 µg/ml is a big difference in comparison. This is due to the Bradford reagent work with Coomassie Brilliant Blue G-250 dye which is a synthetic polyamino acids that binds primarily to basic (arginine) and aromatic amino acid residues but less reacted with other residues. Thus, Bradford reagent is suitable for basic and acidic proteins. The Bradford reagent is suitable for general use, especially for determining the protein content of the cell fraction and assessing protein concentration for gel electrophoresis.
The Bradford assay is a very popular protein assay method because it is simple, rapid, inexpensive and sensitive. The Bradford assay works by the action of Coomassie brilliant blue G-250 dye (CBBG). This dye specifically binds to proteins at arginine, tryptophan, tyrosine, histidine and phenylalanine residues. It should be noted that the assay primarily responds to arginine residues (eight times as much as the other listed residues) so if you have an arginine rich protein, you may need to find a standard that is arginine rich as well. CBBG binds to these residues in the anionic form, which has an absorbance maximum at 595 nm (blue). The free dye in solution is in the cationic form, which has an absorbance maximum at 470 nm (red). The assay is monitored at 595 nm in a spectrophotometer, and thus measures the CBBG complex with the protein. Based on the results in part 1, According to Beer Lambert Law, the amount of light that is absorbed by the solution is directly proportional to the concentration of the sample solution . However from the Graph 1 , most of the points do not align with the best fit line.
There are some reasons may explain why it is happened . Firstly , the clear surface of the cuvette might not clean properly before putting to the spectrophotometer . Next , the solutions might not be mixed properly as the time for each solution mixing in the vortex mixer and it might be caused by pipette the solution inaccurately during preparation . The Bradford assay is faster involves fewer mixing steps, does not require heating, this assay has been found to be useful for peptides and proteins having molecular weights greater than approximately 3,000-5,000, depending on the presence of charged groups. Interfering substances include many detergents and basic buffers; there are modifications of this assay that reduce or eliminate the effects of many interfering substances. The concentration of Bovine Serum Albumin (BSA) of sample A, sample B and sample C were obtained from the Graph 1. The concentration of Bovine Serum Albumin (BSA) of sample A, sample B and sample C are 0.1mg/ml , 0.6mg/ml and -0.28mg/ml respectively . The protein concentration stated in the nutritional information of the apple juice ( sample C ) shown in Figure 3.1 is 0.1g per 100mL . Therefore the concentration of sample C is 1000mg/ml . Calculation for concentration of sample C stated in the nutritional information : 0.1g x 106 = 100000mg
Hence concentration = 100000
The concentration of sample C obtained from the Graph 1 is -0.28mg/ml and it should not show a negative value . It might due to some reasons that are the clear surface of the cuvette was not cleaned properly or the sample was not mixed well in the vortex mixer . As there is a negative readings for sample C , hence the discrepancy between two reading are difficult to be examined .Therefore the two readings of the concentration sample C are assumed to be discrepancy . There is a discrepancy between two readings of the concentration C because the BSA reacts primarily with arginine residues and less so with histidine, lysine, tyrosine, tryptophan, and phenylalanine residues and the BSA assay is less accurate for basic or acidic proteins. Bradford protein assay is less susceptible to interference by various chemicals that may be present in protein samples therefore it is suitable for the analysis of the protein content in food . Bradford protein assay are advantageous compare to the other methods because it is a rapid , simple and sensitive method for estimation of protein concentration in the sample extract . In addition , it colour is virtually complete in 2 minutes and the colour is stable for 60 minutes ( 1 hour ) and it can performed at room temperature . On the other hands the Bradford protein assay is linear over a short range, typically from 0 µg/ml to 2000 µg/ml, therefore the sample solution need to be diluted with distilled water if it’s concentration is beyond 2000 mg/ml .
Protein Assay using Bradford method
Selecting a protein assay
The Bradford Assay