thank you! 4. What are some possible sources of error in regards to the preparat
ID: 195731 • Letter: T
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thank you!
4. What are some possible sources of error in regards to the preparation of the standard curve? 5. Coomassie Blue binds to basic amino acids and to aromatic residues. Since the number of these types of amino acids within a protein determine how much dye will bind to a protein. A Bradford assay is usually operated under the assumption that the unknown protein will have a similar amino acid composition as the standard BSA, but this is not always the case. How would your results be changed if the unknown protein had a higher percentage of basic and aromatic amino acids? Would your results be artificially lower or higher?Explanation / Answer
Basic principle: The suffix "R" in the name of Coomassie Brilliant Blue R-250 is an abbreviation for Red as the blue colour of the dye has a slight reddish tint. For the "G" variant the blue colour has a more greenish tint. The "250" originally denoted the purity of the dye.
The Coomassie blue G250 dye appears to bind most readily to arginyl and lysyl residues of proteins (not to the free amino acids). This specificity of the dye to particular amino acids is the main disadvantage of the dye. Always taking up BSA is not recommendable. It is advisable to choose a protein standard that is likely to give absorbance values close to those for the protein samples of interest . For an example if we are determining the immunoglobulin concentration then IgG can be used as the standard. This assay is less acurate for basic and acidic proteins.
Beyond this IgG is very sensitive to comassie. hence this can also be used as a standard instead of BSA
Question 1: Standard curves are constructed by seeing serial dilutions of a single color of molecule in solvent. They are used to evaluate a spectrophotometer's linearity (straight line in plot) as well as determine the number of molecule (concentration) per ml solvent added to each sample for a given concentration.
Fluorescence is linearly proportional to dye concentration in dilute samples . when the concentration is too great, quenching occurs and the relationship between fluorescence and concentration becomes curvilinear. this shows an error.
Dilution error: accurate pipetting and the proper dilution of the standard accuracy is a must.
control solutions should be made to nullify the spectral variation after each reading.
solutions should be very clear to avoid low absorbance or light scattering specially with tissue and blood samples.
Low fluorescence intensities will introduce error into the flow estimates.
Dye that is susceptible to degrade fast can also result in error.
Staining dyes can also result in error.
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