Affinity purification: Assemble the following components to set up affinity chromatography to purify a protein.Which protein dothe bound protein from the column?youthink is purified in this affinity purification type? Which compound is used to eluteGlutathioneSepharoseColumnBeadSpacer (or arm)GST-taggedprotein

Affinity chromatography: It is a chromatographic technique for the separation of the compound on…

Answered: Affinity purification: Assemble the…

Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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Protein purification table: A 50 ml crude skeletal muscle extract contains 32mg of protein per ml. Ten ul of the extract catalyzes a reaction at a rate of 0.14 µmol product per minute. The extract was fractionated by ammonium sulfate precipitation, and the fraction precipitating between 20% and 40% saturation was dissolved in 10ml. The solution contains 50mg/ml protein. Ten ul of this purified fraction catalyzes the reaction at a rate of 0.65 µmol/min (a) What is the degree of purification (fold purification)? (b) What is the percent yield of the enzyme recovered in the purification?
a. An oligopeptide ALVGALGATPTPQMWSHSWRGVSIKS was digested with trypsin.Which method would be most appropriate for separating the products: ion exchange or gel filtration chromatography? Explain.b. Suppose that the peptide was digested with cyanogen bromide. What would be the optimal separation technique? Explain
Affinity chromatography You have created a fusion protein tagged with Glutathione-S-Transferase (GST). Your lab mate tells you that the affinity columns for this type of tagged protein are very similar to that of Histadine tagged proteins. Using the following elements set up a purification column and construct a protocol for an affinity purification using this tag. A large amount of glutathione is usually used to elute the tagged protein off the column. How might this work?
Purification of a new unknown protein that you isolated from tissue and Assume that you have reached the following data during the characterization; Gel filtration: Gel filtration in protein native conformation When chromatographed, it has a molecular weight of 240000 daltons (240 kDa) is detected to be around. Gel filtration: The same protein is first denatured with 6 M guanidinium hydrochloride subjected to gel filtration chromatography again under denatured conditions. is retained, and the only column from the column with a molecular weight of about 60000 daltons (60 kDa) a protein is obtained. SDS-PAGE: Protein finally SDS-PAGE in the presence of beta-mercaptoethanol (Sodium dodecyl-sulphate polyacrylamide gel electrophoresis) analysis being held. As a result of SDS-PAGE analysis, their weight in the gel is approximately 40000 daltons. Two protein bands corresponding to (40 kDa) and 20000 daltons (20 kDa) is observed. In the light of these findings, the quaternary/quaternary…
topic: Bradford AssayThere are numerous methods of protein determination in use, but this module focuses on the Bradford assay.The Bradford assay is a dye-binding method that employs Coomassie Brilliant Blue G-250, whose structureis shown in Figure 2.3.4.1. Coomassie Brilliant Blue G-250 is a dye that interacts with proteins throughhydrophobic and electrostatic interactions. What are the identities and functions of the components of the Bradford reagent in protein contentdetermination?
When given a purification table that contains the total protein, protein conc., the specific activity and the total activity of 4 different fractions how do you determine which fraction to use for further purification? e.g. do you determine this by the total activity of the fractions? if so would you use the fraction that has the highest total protein activity?
Subject: Biochemistry, chemistry, polyacrylamide gel electrophoresis Differentiate native PAGE and SDS-PAGE in terms of the relative molecular weight of protein bands obtained. Explain your answer.
In Multi-Column Purification of rGFP. What happens to the protein amount, protein purity, and/or specific activity of a purification fraction if one of the three is changed? (i.e. understand the relationship between the three.)
Give two benefits of utilizing the biuret reaction to assess protein concentration over directly measuring protein absorbance at 280 nm.
detail how cation exchange chromatography works and what you would use to elute your target protein. What protein information would you need to facilitate this approach? Would you need to do any protein engineering to utilize cation exchange chromatography, justify your answer?
.A protein gives a single band on SDS gel electrophoresis, as shown in lanes 1 and 2 below. There is little if any effect from adding
Help? Please explain salting out in protein purification.

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