I need help with session 2, number 4. 118 Experimental Biochemistry Table C.2.1.
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Question
I need help with session 2, number 4. 118 Experimental Biochemistry Table C.2.1. Variation of Asso and Initial Rate. A 2s0 Activity (V LDH concentration Specific activity [Pl high low high high high/low low 2. 3. 4. Suppose you wanted to use a protein sample four weeks after storing it at -20° C. Your label might read either "EB/LB-1" or "E. Blake, p. 27, LDH Step 1, 03-05-04, meat extract." Why is the latter label better? List specific advantages by explaining what each item means and why it is important to include. Session 2 1. What is the stationary phase in an affinity chromatography column? What is the mobile phase? 2. What are the purposes of the low NADH and high NADH washes through the column? 3. Explain why you measure the absorbance at 280 nm to monitor the progress of the affinity chromatography column. Which type of amino acid residues contribute to Agso? Name them and explain why. dgnore NADH, which contributes but is not the focus of this question.) How does Aso depend on the purity of the protein preparation? 4. Table C.2-1 shows three ways in which the measured Ass and initial rate might vary with a. How large is the protein concentration, [P], in each of the three cases (in qualitative respect to each other. terms)? b. How are V, and [P] used to calculate the specific activity? What are the units? c. Explain what you can conclude about [P) and the specific activity of the preparation in each of the three cases.
Explanation / Answer
4. Letter levelling is important on tubes containing proteins in lab, so it can be easily read, which protein you are using out of multiple proteins in the freezer. Along with this extraction date should be mandatory to be mentioned on tube.
Session-2
1. Affinity chromatography is very specific chromatography. It is used to separate various molecules like antigen/antibody, enzyme/substrate, receptor/ ligand etc. For isolation of one molecule out of give pairs (Antibody/antigen), other can be attached with solid phase. For example: Stationary phase is mostly gel like agarose coated with antibody and mobile phase can be any solution containing antigen.
2. To isolate high affinity molecules low NADH will be used for elution whereas for less affinity molecules high NADH concentration will be needed.
3. In affinity chromatography, especially when we are dealing with proteins, we use to measure absorbance of ultraviolet radiations at 280 nm (A280). Aromatic amino acids (Tyrosine, tryptophan and phenylalanine) in proteins give absorbance at 280. Tryptophan is responsible for most of the absorbance of ultraviolet light at 280 nm.