Introduction:
In 1976, Marion Bradford designed the Bradford method that is widely used in measuring the concentration of protein in a solution. When Coomassie Blue is added to a solution containing protein, the solution colour changes from brown to blue and this is attributed to the formation of a protein complex. The concentration levels of protein can therefore be measured by observing the dye color, which fully absorbed at a certain wavelength. The standard curve is used to calculate the concentration of protein because it represents the known BSA (bovine serum albumin) dilution. (Harvard, 2012)
A study by Daiet (2008) analysed murinae in relation to the SLE model of CGVHD, this was done by injecting murinae with lymphocytes and the production of albuminuria was assessed using Bradford assay test. Antibody presence was determined using ELISA test. Servat (2007) used the ELISA test to analyse dog vaccine effectiveness by using this method to analyse the antibodies of rabies. (Smooker, 2010)
This practical report assesses non-transformed and transformed E coli β-galactosidase protein utilising the Bradford assay and the Elisa assay. Protein concentration was determined using the Bradford assay test while the presence of β-galactosidase protein was determined using the indirect ELISA assay. Sonication was used in both cases in order to extract protein, DH5α E Coli strain was used, and p Blue script vector was used to transform this sample. (Piercenet, 2011)
1. SLE- systemic lupus erythematosus. 2. CGVHD- chronic graft versus host diseases.
3. ELISA- enzyme linked immunosorbent assay
Methods and materials:
The method used and materials used are as per the practical manual.
Results:
Bradford assay:
This method was used to determine the protein levels; duplicate and blank absorbance readings were recorded. The mean absorbance level was determined and this value subtracted from the blanks in order to come up with the standard curve. This curve can then be used to determine E. coli protein content. The table below indicate the protein levels and mean absorbance:
Table 1: mean absorbance (nm) and protein levels (ug)
Mean of Duplicates
Mean-Blank
Protein Content (µg)
Graph 1: protein levels versus absorbance
Y axis = Absorbance
X axis = Protein Level
Y = 0.027x
Indirect ELISA:
Transformed strain
Absorbance-Blanks Mean
log titre
Graph 2
Non transformed strain
Absorbance-Blanks Mean
log titre
Graph 3
Discussion:
Protein levels were determined using the BSA curve; the values were then used to determine the samples to use in the ELISA method. This method was to help determine the presence or absence of E coli protein. Graph 1 and graph two show a logarithmic graph representing the transformed and non-transformed strain. These graphs help to determine the optimal concentration levels for antibodies and antigens. The optimal level is determined by inserting a perpendicular line drawn from the midpoint of logarithm trend line. The two graphs can then be used to determine the optimal levels and in this case the optimal level.
The above discussion focuses on the Bradford method introduced by Marion Bradford in 1976; it is used in measuring the concentration of protein in a solution. The concentration levels of protein can therefore be measured by observing the dye colour, which is fully absorbed at a certain wavelength. In this paper, the standard curve is used to calculate the concentration of protein because it represents the known BSA (bovine serum albumin) dilution.
Conclusion:
It is evident that the Bradford and the ELISA methods play a major role in the determination of protein and antibodies in solutions. It has been widely used and most notably by researches like Daiet (2008) who used Bradford assay method to establish the rat and mice SLE model of CGVHD, the researcher then used the ELISA test to determine the presence of Antibody in the solution. Servat (2007) also used the same method to determine the effectiveness of rabies vaccine, the ELISA method was used to check for antibodies in the solution.
The sonication process has worked in this test; this is because the absorbance data for both the transformed strain and non-transformed strain are different as indicated by the graphs. The results indicate an over expression of β-galactosidase protein in the transformed train, this means that these two test can be used together to make conclusion about the effectiveness of a solution, example new vaccines as show in the results.
Reference:
Harvard. 2012. Bradford Protein Assay. Retrieved 28th August, from:
Piercenet, 2011. Overview of ELISA. Retrieved 28th August, from: http://www.piercenet.com/Proteomics/browse.cfm?fldID=F88ADEC9-1B43-4585-922E-836FE09D8403
Smooker, P. 2010. Advanced Immunology and Cell Technology Practical Manual. Melbourne: RMIT University.
