Here's a transcription of the text from the image:---7. Imagine you're purifying a protein with a pI of 6.5 that has a 6-Histidine tag from *E. coli* lysate. You're planning to use Ni-NTA resin (nickel resin), which has a high affinity for the 6-His tag, to purify your protein from the rest of the lysate. 1. Which pH ranges would you expect a protein with a pI of 6.5 to be stable in? (Remember: pI is isoelectric point) 2. What pH range is recommended by Ni-NTA resin manufacturers for purification? 3. What pH do you think would work best for your purification process and which buffering agent(s) would work well for that pH?8. A lab mate prepared a 50 mM Tris buffer for you, and told you that it was pH 8. But the next day, you took the buffer out of the refrigerator and...---Explanation of the context:- The text pertains to protein purification using a 6-His tag and Ni-NTA resin, focusing on appropriate pH conditions for stability and purification efficiency.- It involves scientific concepts such as the isoelectric point (pI) and buffering systems.- Ni-NTA resin is typically used in affinity chromatography to isolate histidine-tagged proteins.- Tris buffer is commonly used in biological labs for maintaining pH during experiments.

Ni-NTA agarose is an affinity chromatography matrix that is used for the purification of recombinant…

Answered: 7. Imagine you're purifying a protein…

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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Using a detailed scheme, propose a step-wise protocol to purify protein B by ion exchange chromatography (Explain your logic/choices).
Modify isoleucine to show the predominant forms at pH 1, 7, and 13. Isoleucine has p?ap�a values of 2.4 (carboxyl group) and 9.7 (amino group).
calculate the volume of stock solutions required to make up the buffer solutions that will be used for protein purification. The solutions you need to prepare for purification are: i. Binding Solution A: make up 50 mL 50 mM HEPES buffer (pH 7.5), 300 mM NaCl, 5mM imidazole, 5% (v/v) glycerol ii. Wash Solution B: make up 50 mL 50 mM HEPES buffer (pH 7.5), 300 mM NaCl, 75mM imidazole, 5% (v/v) glycerol iii. Elution Solution C: make up 10 mL 50 mM HEPES buffer (pH 7.5), 300 mM NaCl, 500 mM imidazole, 5% (v/v) glycerol please show your working . Thnk you
Assume a pH of 7.0 in aqueous solution. Please draw a tripeptide containing (in this order) a residue with a residue with an non-polar sidechain containing more than five carbons, a positively (+1) charged sidechain, and a residue with a polar (but not charged) sidechain with more than two carbons. Please give the names of the amino acid residues that you drew.
1. Draw the tetrapeptide Met-Ala-Thr-Thr at a ph of 7? 2. Draw the tetrapeptide Met-Ala-Thr-Thr at a ph of 12?
You are given a protein solution with a concentration of 0.15 mg/ml. Suppose that we want to prepare a solution containing 100 μg of the protein at a concentration of 1 mg/ml. To achieve this, we will first dry down enough protein solution to obtain 100 μg of proteins. How much solution do we need for drying down? How much volume of H2O do we need to add to the dried protein to obtain the desired concentration?
Consider the phenolic hydroxyl group of a particular Tyr residue in a protein. Suppose the hydroxyl group in the unfolded protein in aqueous solution, where the group is exposed to H2O, has a pKa of 10.0. If that group is found in a hydrophobic environment in the interior of the protein when the protein is folded into its native tertiary structure, would you expect the pKa of the phenolic hydroxyl to be higher or lower in the folded protein interior than in H2O? Explain your reasoning.
The following proteins were separated by SDS-PAGE in the presence of mercaptoethanol. Sketch the relative positions of the various polypeptides on the gel. Label the positive and negative ends of the gel.Protein A: 40 kDa single polypeptideProtein B: 80 kDa protein, made up of two subunits of molecular weight 20 kDa and 60 kDa, held together by noncovalent interactionsProtein C: 200 kDa protein, made up of four identical subunits (50 kDa each) linked together by disulfide bonds
a) The pl of a protein is 9.0. What will be the net charge (positive, negative or neutral) on the protein under the following conditions: A buffer with pH 9.0: A buffer with pH 7.0: A buffer with pH 10.0: b) Your protein is present in a sample buffer which has a pH 7.0. The sample is applied to an ANION EXCHANGE column. Will protein 1 bind to the beads on the column? Yes/No
A schematic diagram of the helical structure of cytochrome b562 is reproduced below. Thisprotein belongs to the family of -proteins that have a four-helix bundle. Number the helices 1 – 4according to their N  C direction. Indicate relative orientations of the macrodipoles of helices 1 – 4adjacent to the diagram of cytochrome b562. Remember that according to the definition of a dipolethat the arrow points towards the positive end.
Suppose you have a solution of a protein, which contains a specific Tyr residue that has an actual (measured) pKa of 8.8. The protein binds a ligand by several noncovalent interactions, one of which is a hydrogen bond in which the Tyr phenolic hydroxyl group must serve as a hydrogen bond donor. Calculate the percentage of the protein molecules in which that tyrosyl residue's phenolic hydroxyl group could serve as a hydrogen bond donor at pH 8.5
+H₂N-CH-COO™ T CH₂ I CH₂ 1 CH₂ I +H₂N-CH₂ A. O amino acid C amino acid D O amino acid E amino acid B +H₂N-CH-COO™ I CH₂ I CH₂ 1 O amino acid A C=0 B. If the pH decreases enough, this amino acid will become protonated and could contribute to the denaturation of the protein. +H₂N-CH-COO™ 1 CH₂ I OH C. +H₂N-CH-COO™ CH H₂C CH3 D.

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