Protein estimation by Bradford’s method Tutor name November 2, 2012 Abstract Proteins are essential body components and are fundamental to biochemical processes in the body. They for example form the basis of biochemical reactions through enzymes…
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This paper applies the Bradford’s method to investigate the relationship between protein concentration and absorbance rate. The study is based on established assumption that observed rate of absorption is proportional to protein concentration and developed relationship between known concentrations and observed absorbance rates can be used to determine unknown concentrations, given their absorbance rates. The study’s data identifies a deviation from the proposed proportionality between concentration and absorbance rates. Significance of this inconsistency is ascertained by analysis of variance that adopts the null hypothesis for lack of significant relationship between concentration and absorbance rates. This is because of the high probability value, 0.056 percent. Application of the proposed regression model confirms the inconsistency by yielding a negative concentration. The paper therefore concludes that the inconsistency with established literature is because of inaccurate experimental results. Protein estimation by Bradford’s method Introduction Proteins are essential components of cells and organs, a factor that forms the basis of their significance in the body as well as the need for their intake in nutrients to meet required levels for a healthy body. Nutritional needs institute the importance of developed knowledge of required protein intake levels, and determination recommended quantities of food supplies that can meet such required levels. Existence of protein in definite concentrations in body fluids also identifies the significance of studying protein concentration in the body, which can be used to determine normalcy or existence diseases to alter equilibrium protein concentrations (Chem, p. 105). There are a number of techniques, such as the Bradford method, which can be used to determine protein concentrations in compounds. The method applies spectroscopy to relate known concentrations to their corresponding absorbance rate. This is because of a linear relationship between concentration of protein in a solution and the ability of that solution to absorb dye (Maud and Foster, p. 164). Visibility of the absorbed dye, which is proportional to the dye’s concentration in the solution, is therefore used to establish a relationship between absorbance readings from the spectroscopy and protein concentration (Ruf, p. 1). The Bradford’s method further assumes that protein concentration is the only determinant of the dye’s absorbance. This means that for any given environmental conditions, similar protein concentrations yield similar absorbance rates (Thermo, p. 1). A linear regression model can therefore be developed and used to determine unknown concentrations, given their corresponding absorption alues. The regression model determines existence of a significant relationship between the known concentrations and the absorbance rates for predictions, and regression coefficient (Ross, p. 131- 134). This study was performed to ascertain existence of a relationship between known protein concentrations and their corresponding absorbance rates. The study aimed at using the established relationship to determine unknown protein concentration, based on its absorbance rate. In order to achieve its objective, the study explored the question, ‘is there a significant relationship between protein concentration and absorbance?’ The study investigated the following set of hypotheses for the research question. H0: There is no significant
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Bradford assays are among the methods used to determine the concentration of protein, relative to a standard. This technique is based on the formation of a complex between the dye, Brilliant Blue G and proteins in solution. The complex being formed causes a shift in the absorption peak from 465nm to 595nm.
Fig 1. Fluorescence intensities of a) tyrosine b)Tryptophan at and c) Thioflavin T at 25 0c and three different pH values. The fluorescence intensities for these three were measured at 303, 348, 482 nm respectively, in all the experiments.
There was slight increase in Tryptophan fluorescence at pH 5.0 and 7.0.
This report examined the features of E coli in respect to the expression systems with an emphasis on the limitation number that have been addressed. For a high gene dosage to be achieved, the heterologous cDNAs are normally cloned into replicate plasmids in a fashion that is relaxed and are normally existence at 15-60 copies per cell.
he template strand of DNA, which contains a nucleotide sequence encoding for the protein identified in the answer to question 3c from the Transcription activity above:
Glycine, methionine, valine, glutamic acid, glutamine, cycteine, cysteine, alanine, serine, valine, cysteine,
With respect to their structure, biochemists often refer to four different aspects:
Secondary structure, local interactions held together by hydrogen bonds between the lone pair of electrons of an oxygen atom and the hydrogen attached to a nitrogen atom.
It was also observed that nutritional requirements for the vital minerals of calcium and iron were often lacking in our diet and the need to take a vitamin supplement is suggested.
A comparison of three methods for the estimation of dietary intake was made using the 7-day Weighed Record, 24 hour Recall, and Duplicate Diet Methods.
4. If a particular protein was absent, it would lead to errors in protein synthesis. Errors in protein synthesis disrupt cellular fitness, cause disease phenotypes, leads to loss of function of the protein (non-functional proteins) and protein misfolding.
After the 30 minutes of incubation the rank is removed from the water bath and the samples allowed to cool.1 ml of each reaction is transfered to a 1 ml cuvette and A562 in a spectrophotometer read. The spectrophotometer zero is set to
Proteomic is generally defined as the direct analysis of proteins in terms of their presence and relative abundance. Gel electrophoresis is a significant methodology employed for extraction of proteins in proteome analysis. The most commonly used technique in gel electrophoresis is Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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