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explain how you would control for interference in a protein assay?.
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- Why is it not necessary to dilute your protein samples with buffer in a bradford protein assay experiment?At higher amounts of protein, the Bradford assay is not linear. Consider the plot to the right: what is the maximum amount of protein a sample could contain and still fall within a standard curve? Briefly explain your reasoning.Mass spectrometry is a powerful tool in proteomics. What are the four key features of a mass spectrometer? Describe briefly how MALDI and two-dimensional polyacrylamide gel electrophoresis could be used to identify a protein expressed in cancer cells but not in normal healthy cells.
- How would you determine the concentration of a protein that falls outside (above or below) the linear range of this assay? asapDescribes a method known as Western blotting that can be used to detect a polypeptide that is translated from a particular mRNA. In this method, a particular polypeptide or protein is detected by an antibody that specifically recognizes a segment of its amino acid sequence. After the antibody binds to the polypeptide within a gel, a secondary antibody (which is labeled) is used to visualize the polypeptide as a dark band.For example, an antibody that recognizes α-galactosidase A couldbe used to specifically detect the amount of α-galactosidase A proteinon a gel. The enzyme α-galactosidase A is defective in individuals with Fabry disease, which shows an X-linked recessive pattern of inheritance. Amy, Nan, and Pete are siblings, and Pete has Fabry disease. Aileen, Jason, and Jerry are brothers and sister, and Jerry has Fabry disease. Amy, Nan, and Pete are not related to Aileen, Jason, and Jerry. Amy, Nan, and Aileen are concerned that they could be carriers of a defective…What assay uses the fact that activators can be separated into DNA binding and activating domains? What information do you gain from this assay? Describe briefly how to perform this assay.
- Describe the p-nitrophenol assay and how a standard curve can be used to calculate the concentration of an unknown .In selecting target cells to receive a transferred gene in gene therapy, what factors do you think would have to be taken into account? Explain your answer.Sketch how an ion-selective field effecttransistor (ISFET) can be used as a biosensor to detect DNA in solution,detailing the mechanism for sensing. Explain why the use of a SiNW could leadto increased sensitivity?
- Biochemistry: Site-directed mutagenesis, in which individual amino acid residues are replaced with others, is a powerful method to study enzyme mechanisms. In experiments with particular enzyme, various lysine residues were replaced with aspartate, yielding the results summarized in the table below: Enzyme Form: Enzyme Activity (U/mg) Native enzyme: 1,000 U/mg Recombinant Lys 21 to Asp 21: 970 U/mg Recombinant Lys 86 to Asp 86: 100 U/mg Recombinant Lys 101 to Asp 101: 970 U/mg a. What might be inferred about the role of Lys 21, 86, and 101 in the catalytic mechanism of this enzyme? b. Discuss where within the enzyme one might find Lys 21 and 101. Are these residues likely to be evolutionary conserved in this enzyme? Explain c. Is Lys position 86 likely to be evolutionary conserved? ExplainDiscuss the following statement: “from the nucleotide sequence of a cDNA clone, the complete amino acid sequence of a protein can be deduced by applying the genetic code. thus, protein biochemistry has become superfluous because there is nothing more that can be learned by studying the protein.”Describe the rationale behind the electrophoretic mobility shift assay.