A researcher is trying to purify a protein that needs to be in a reducing environment and wants to use IMAC to purify their protein. Specifically, he wants to use dithiothreitol (DTT) at high concentrations to maintain a reducing environment. When he purifies hisprotein using Ni-NTA, he has no problems with his protein binding to the column. However, when he uses another column, Ni-IDA, most of the protein doesn't bind. He discovers that the IDA resin only coordinates Ni²+ with 3 three ligands where NTA is known tocoordinate with 4 four ligands. How do you suspect the DTT is making the Ni-IDA ineffective at binding a 6xHis tagged protein (hint: it may help to draw the structure of DTT)?

let's delve deeper into why DTT may interfere with the binding of the 6xHis-tagged protein to the…

Answered: A researcher is trying to purify a…

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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You have been given an unknown low weight polypeptide to sequence. Luckily, you have a plentiful supply. You are given a limited amount of Edmond's reagent-just enough to perform 1 restricted digestion for every fragment that you generate but not for complete digestion of every fragment. You have the following enzymes and reagents available to do your digestion analysis: Chymotrypsin, V8, HCI (1M), Cyanogen Bromide (CnBr) and 50mM of ammonium bicarbonate. You are only given enough Carboxypeptidase for one fragment analysis. You fortunately have a plentiful supply of ion chromatography columns, eluting buffers, and SDS-PAGE gels to help you separate individual amino acids for analysis and fragment size (length) determination. And you have a really good spec and an HPLC. From the information given below, you will need to construct the sequence of your peptide! You also have the following information: 1. Digestion of the intact polypeptide with 1M HCl will liberate all amino acids (you…
Jacqueline isolated an interesting new protein from the broth of a yeast culture. After purification of the protein, she requested an amino acid analysis and found that the protein contained 0.57% tryptophan by weight (tryptophan Mr-204). Calculate the minimum molecular weight of the protein (i.e., assuming there is only one tryptophan residue per protein molecule).
This problem concerns a "typical" tyrosine residue found on the surface of a globular protein. a) If the pKa of this residue is 10.1, at what pH will this residue be found in its deprotonated state in 99% of protein molecules? b) How would the presence of a neighboring negatively charged residue effect the equilibrium expression for the dissociation of tyrosine? c) Would the presence of the negatively charged residue raise or lower the pKa of this tyrosine? Justify and/or explain your answers in 1–2 sentences each for parts b) and c).
You start with 100 units of protein activity and 100 grams of total protein. After the first centrifugation step, you have 25% less activity and 85% less total protein. What is your fold-purification of the target protein? 0.3-fold 3.4-fold 75-fold 5-fold 20-fold
A protein has been sequenced after cleavage of disulfide bonds. The protein is known to contain 3 Cys residues, located as shown here. Only one of the Cys has a free —SH group, and the other two are involved in an —S—S— bond. The only methionine and the only aromatic amino acid (Phe) in this protein are in the positions indicated. Cleavage of the intact protein (i.e., withdisulfide bonds intact) by either cyanogen bromide or chymotrypsin does not break the protein into two peptides. Where is the —S—S— bond (i.e., AB, BC, or AC)?
You have isolated from a rare fungus an octapeptide that prevents baldness and you wish to determine its amino acid sequence. The amino acid composition is K2, D, Y, F, G, S, A. Reaction of the intact peptide with dansyl chloride yields dansyl-A. Cleavage with trypsin yields peptides whose compositions are: (K, A, S) and (G, F, K) plus a dipeptide. Reaction with chymotrypsin releases free D, a tetrapeptide with composition (k, S, F, A) and a tripeptide whose composition following acid hydrolysis is (G, K, Y). The enzymatic digests are each carried out on the whole, undansylated peptide.What is the sequence?
Make a table with a scale of absorbance and the concentration of protein in Chromatin sample from the following data for excel graph Absorbance=660nm following data are of tubes with concern A =0 B=0.036 C=0.011 D=0.001 E=0.027 F=0.020 G=0.032 H1=0.176 H2=0.183 I1=0.150 I2=0.171 also plot the graph??
1) In MALDI-TOF data, when performing peptide mass mapping search, a) The number of peptides matched decreases from 13 to 11 and the sequence coverage decreases to 47%, but the protein score increases from 111 to 124. Explain. b) The nnumber of 11 peptide masses matched and the sequence coverage is 47% (same as in part a), but the protein score increases to 157. Explain.
When sequencing proteins, one tries to generate overlapping peptides by using cleavages at specific sites. Which of the following statements about cleavages caused by particular chemicals or enzymes are true? Cyanogen bromide cleaves at the carboxyl side of threonine. Chymotrypsin cleaves at the carboxyl side of aromatic amino acids. Trypsin cleaves at the carboxyl side of Lys and Arg. Chymotrypsin cleaves at the carboxyl side of aspartate and glutamate.
You gathered your data from Lab 4, and found the following values: Total Activity of lysozyme in HEW: 14 units Total Activity of lysozyme in carb 1: 21 units Total amount of protein in HEW: 70 mg Total amount of protein in Carb 1: 15 mg Calculate the extent of purification of lysozyme in Carb 1. 150% 7 fold 70% 15 fold
What would the order of migration be (bottom to top in the gel) in a SDS-PAGE for the following proteins? Concanavalin A (ConA) (a homotetramer of 106 kDa), lysozyme (a monomer of 14.3 kDa) & horse liver alcohol dehydrogenase (ADH) (a homodimer of 80 kDa). a. ADH, ConA, lysozyme b. ConA, ADH, lysozyme c. Lysozyme, ADH, ConA Od. Lysozyme, ConA, ADH
1. A) Explain this statement, “Most enzymes are gene-encoded proteins, but most enzymes are not only protein". B) You recently isolated a new enzyme in your lab, and you named it "lipidase 1120". As a temporary name, this is fine. Who is responsible for assigning a formal name, and on what criteria will that name be assigned?

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