Chapter 5 Protein Purification HW +RQ

The Edman degradation uses to label the N-terminal protein, whereas the Sanger method modifies the N-terminal residue of a peptide with _____________ .

1. phenylisothiocyanate 2.1-fluoro-2,4-dinitrobenzene

Congratulations, you have purified your protein of interest (YPOI). You and your research partner have subjected your protein to polyacrylamide electrophoresis. You treated your protein with SDS and a reducing agent (beta-mercapto ethanol) and boiled it. Your partner ran a native gel without the SDS and reducing agent treatment. You both observe one band on each gel, but your protein (denaturing SDS PAGE) migrated as a 23,000-dalton protein, while your research partner's gel showed a band at 92,000 daltons.After some discussion, you feel that your protein is a and that the SDS and reducing agent have:

1. tetramer 2. denatured the protein, which is made of equally sized monomers.

Western blotting is used to identify specific proteins from a mixture of proteins separated by polyacrylamide electrophoresis. Label the steps of Western blotting in the figure.

1. transfer proteins from a gel elctrophoresis to a filter membrane 2. Block the membrane with a non-specific non antibody binding protiein 3. Incubate the primary antibody with the membrane 4. Incubate the membrane with a conjugated secondary antibody 5. The antibody complex containing the conjugated enzyme is used to detect the antigen protein

Which of the following is correct concerning protein structure determination by NMR spectroscopy?

NMR provides dynamic information about protein structure. NMR studies require highly concentrated solutions of purified proteins, which can be a challenge for some proteins. Some properties can be studied for very large proteins, but full structure determination is limited to proteins smaller than 100 kDa. The distances between atoms are only approximate and are not exactly known. However, NMR is useful for studying protein dynamics and conformational changes.

Someone who is allergic to a particular compound (allergen) will often create a subtype of antibodies (immunoglobin E) to the allergen, and histamine levels will increase to produce the immune response. Although testing a particular food for the allergen is relatively straightforward, learning which potential allergen caused the response is much more challenging. Which might be the best technique to determine which allergen a person has been exposed to, and why?

ELISA - using an allergen in a sandwich ELISA will capture the antibodies produced against the allergen.

You have discovered a small organic compound that you think will cause a significant shift in the peptide loop covering the active site of the enzyme, inhibiting the protein's function. Which approach(es) would be most appropriate to test for this hypothesis?

NMR spectroscopy X-ray crystallography

Which of the following is FALSE when isolating proteins from whole cells? The amount of protein in each fraction isolated during centrifugation separation increases. Cells can be homogenized using a French press. Salting out separates proteins based on their solubility. The amount of protein in a fraction is measured by reading the sample absorbance at 280 nm.

The amount of protein in each fraction isolated during centrifugation separation increases.

Which of the following is NOT correct when B cells produce antibodies? Each B cell only makes a single type of antibody. Two classes of immunoglobulin light chain are produced. Five classes of immunoglobulin heavy chain are produced. The antigen binds to structured loops in the constant domains.

The antigen binds to structured loops in the constant domains. B cells produce two classes of light chains and five classes of heavy chains and each B cell only makes a single type of antibody. Antigen binding is via flexible loops in the variable regions of the light-chain and heavy-chain polypeptides.

Adding highly antigenic peptides, called epitope tags, to the amino or carboxy terminus of a protein enhances the ability of commercially available antibodies to be used for Western blotting and immunoprecipitation. Which of the following best describes how epitope-tagged proteins are produced?

The gene encoding the protein of interest is modified to include a short sequence of amino acids that is not commonly found in proteins.

What protein feature will cause the protein to bind to a metal in chelation affinity chromatography—specifically, a resin with immobilized Ni2+?

recombinant proteins engineered to have six or more histidines at the N or C terminus of the protein

The mass-to-charge ratios of denatured proteins are equivalent for different mass proteins. However, the cross-linked nature of the acrylamide media can limit migration through the polymer matrix. Gels with less cross-linked acrylamide (low % SDS gels) will do which of the following?

separate larger proteins at the expense of smaller proteins, which will not resolve well

With proteins being separated via gel electrophoresis,

smaller proteins move faster. Gel electrophoresis separates biomolecules, like proteins, based on charge and size. Smaller proteins will move faster than larger proteins because they can maneuver more easily through the matrix. Molecules move toward the anode, which means more negatively charged proteins will move faster than positively charged proteins.

Which of the following statements best describes immunoprecipitation?

use of high-affinity antibodies linked to a dense carbohydrate bead to isolate protein antigens

There are five steps to generating a peptide using solid phase synthesis. Place the appropriate description at each step.

(top right)---> deblocking of residue attached to resin (top left)--> activation of Fmoc-blocked residue (middle 1)--> coupling of amino acids (middle 2)--> final deblocking of peptides with the synthesized amino acids (middle 3)--> cleavage from resin and deprotection of all amino acid side chains

The Edman degradation is able to sequence up to 50 residues using a simple method. Place the description for each step in order of the Edman degradation technique.

1. An unlabeled peptide is mixed with phenylisothiocyante to covalently bond 2. Peptide is treated with trifluoroacetic acid to cleave the bond between the 3. The PITC-labeled amino acid is extracted using an organic solvent and further modified 4. Using standards, paper chromatography identifies the modified amino

Gel filtration

1. Can be used to determine the molecular mass of an unknown protein using a set of proteins with known molecular mass 2. Uses a porous bead made of complex carbohydrates that allow proteins to go through or around 3. Small proteins are retained due to a longer pathway through the column. 4. Two proteins of the same size but different shape (globular vs fibrous) can be separated by this method.

ESI

1. Degrades and charges the peptide with high voltage 2. Sample becomes ionized due to high voltage. 3. Ionization evaporates solvent, leaving peptide in gas phase to travel through the separating mass spectrometer.

Ion-Exchange

1. Exploits the charge state of a protein to adhere to a chromatography resin 2. DEAE is a positive charged resin used in this type of chromatography. 3. Proteins are displaced from the resin by changing pH or increasing the ionic strength (salt conc.) of elution buffer.

MALDI

1. Laser will be absorbed by the peptide containing material releasing the peptide into the gas phase. 2. Proteins or peptides are mixed in a solid matrix rather than a solution. 3. Peptides become fragmented and charged due to laser exposure.

__________________________________ provides information on the structure of a purified protein based on its nuclear spin properties in a magnetic field, whereas _________________________________ uses diffraction pattern of proteins in solid phase.

1. NMR spectroscopy 2. X-ray crystallography

X-ray crystallography

1. Nearly any sized protein can be used for this type of structural analysis. 2. Dynamic changes are not easily captured. 3. Poorly ordered domains, including disordered domains, will have mulitple arrangements averaging out a signal.

NMR spectroscopy

1. Requires isotopic labeling of elements such as N, C, or H 2. Smaller proteins are more amenable for this type of structual analysis. 3. Large proteins will reorient slowly in solution, averaging out the signal.

Affinity

1. Separates proteins based on specific binding properties of the target protein 2. Natural or mimicked ligand of the target protein is bound to a solid-phase resin, often a dextran bead. 3. Antibodies are sometimes used to bind target proteins in this chromatography. 4. Metal ions can be fixed to a resin and bind to specific sequences of amino acids such as histidine.

ESI and MALDI

1. Uses mass-to-charge (m/z) ratio to determine molecular mass 2. Can be used for proteins or peptides that have been treated with proteases like trypsin 3. Is based on the mass and acceleration though a chamber ending in a detector

You want to test a new drug that inhibits an important disease-fighting protein kinase involved in plant defense against mold. You think that several proteins will not be phosphorylated when the plant is treated with the drug and Cy5. To test this, you add the drug to a plant extract and leave a second extract alone with added Cy3 (the control). To see how many proteins changed by the drug, you perform a 2-D DIGE analysis. Phosphorylation will the overall charge state of the protein. Any proteins affected by the kinase inhibitor will have a ________ spot on the 2-D DIGE gel, whereas proteins not altered by the drug will have a ______ spot.

1. decrease 2. red 3. green

The enzyme-linked immunosorbent assay (ELISA) uses antibodies to detect specific antigens. Label each of the indicated components of a "sandwich" ELISA.

1. horseradish peroxidase= small green pea 2. light orange bar on red and orange Y= horseradish peroxidase-linked antibody 3. Purple bar on purple and blue Y= detection antibody 4. divit in big green blob for detection antibody=Epitope 2 5. Big green blob= antigenic protein 6. divit on bottom of antigenic protei epitope 2 7. pink bar on pink and orange Y= capture antibody

Which process in solid-phase peptide synthesis is being depicted in Step 2 of the figure below?

Activation Solid-phase peptide synthesis involves the coupling of animo acids to form the desired peptide. In step 1, the immobilized amino acid is deblocked, which removes the Fmoc protecting group. In step 2, the incoming amino acid is activated using DCC. Step 3 is the coupling step, where the two amino acids are joined to form a peptide bond. These steps are repeated until the final amino acid is added. Step 4 is the final deblocking step and step 5 involves cleavage from the resin and deprotection of the amino acid side chains.

Which of the following sample preparation steps is used for both native PAGE and SDS-PAGE?

Addition of glycerol to the sample loading buffer SDS-PAGE uses denaturing conditions to separate polypeptides on the basis of size. Native PAGE uses non-denaturing conditions to maintain protein structure and allows for characterization of protein complexes. Sodium dodecyl sulfate, or SDS, is a detergent that denatures proteins and gives the protein an overall negative charge. β-mercaptoethanol reduces disulfide bonds. Heating the sample facilitates denaturation. Glycerol in the sample loading buffer gives density to the sample so it sinks to the bottom of the gel well when loading.

Immunoprecipitation is a variation of what type of protein purification method? Gel filtration chromatography Affinity chromatography Ion exchange chromatography You Answered Gel electrophoresis

Affinity chromatography Immunoprecipitation is a form of affinity chromatography. Instead of a small molecule ligand, an antibody is immobilized on a solid support and used to capture a protein or protein complex of interest. Immunoprecipitation is useful for isolating proteins prior to mass spectrometry analysis.

On the 2-D gel shown in the figure below, where would a protein with a high pI and a high mass be found?

C (top right) In 2-D gels, proteins are first separated based on their isoelectric points (pI) in an isoelectric focusing experiment. The pI-separated proteins are then separated again on the basis of mass in a standard SDS-PAGE gel. Thus, low pI proteins are on the left and high pI proteins are on the right, while high mass proteins are at the top and low mass proteins are at the bottom of the gel.

Which resin should you subject your mixture to get the best separation? Choose the resin with the correct attribute of the resin and protein that will separate the proteins. CMC cellulose, because the pH is above two of the contaminating proteins and less than YPOI and contaminant protein 3. These two proteins can then be separated by ionic conditions (salt gradient). B. gel filtration, because the proteins are all within the exclusion range of this resin C. CMC cellulose, because all four proteins will be positively charged and can be separated by ionic conditions during elution D. DEAE cellulose, because the proteins will be charged and can be separated by ion exchange chromatography

CMC cellulose, because the pH is above two of the contaminating proteins and less than YPOI and contaminant protein 3. These two proteins can then be separated by ionic conditions (salt gradient).

Which of the following specifically cleaves methionine residues?

Cyanogen bromide Many reagents used to cleave polypeptides are protein proteases, which target certain amino acids. Cyanogen bromide is a chemical reagent that reacts specifically with methionine to cleave the peptide backbone.

You have a collaborator who thinks she has created a compound to bind and inhibit an enzyme involved in lung cancer metastasis. Which of the following experiments using lysate from a tumor should be considered to test whether or not this drug would bind to the protein?

Fix the drug compound to a chromatography bead and perform an affinity chromatography on the lysate to detect the protein that binds the drug.

Which of the following procedures is NOT used for polyclonal antibody generation? Immunization of animal with antigen Isolation of blood serum from immunized animal Fusion of B cells with immortalized tumor cells to form hybridoma Affinity chromatography to isolate desired antibodies

Fusion of B cells with immortalized tumor cells to form hybridoma Polyclonal antibodies are a mixture of antibodies generated in response to an antigen. Immunizing an animal will result in antibody production, so the blood serum can be isolated and purified via affinity chromatography to isolate the collection of antibodies that bind to the antigen. This process can be used to generate polyclonal antibodies until the animal dies. Hybridomas are used for monoclonal antibody production.

In the figure below depicting a Western blot experiment, which step is depicted in C?

Incubation and binding of the secondary antibody In Western blotting, proteins are transferred from SDS-PAGE to a nitrocellulose membrane. The membrane is washed with blocking buffer to prevent non-specific antibody binding. An antibody specific for the protein of interest, called the primary antibody, is introduced and allowed to bind to the protein. A second antibody that recognizes the first antibody and is linked to an enzyme is then introduced. Addition of the enzyme substrate results in a detectable product that indicates the location of the protein of interest. (one thing)

When using tandem mass spectrometry for peptide sequence determination, what is the input into the second mass spectrometer from the collision chamber?

Isolated and fragmented peptides Tryptic fragments are introduced into mass spectrometer 1 via electrospray ionization. These fragments are separated by size and a narrow range of masses is selected to enter the collision chamber, where the peptides are fragmented into smaller pieces. These fragmented peptides then enter mass spectrometer 2. The resulting fragments are detected, analyzed, and compared to peptide masses in databases to determine the peptide sequence.

Which of the following amino acids would not be cleaved by chymotrypsin?

K Proteases are often used to cleave polypeptides prior to Edman degradation. Chymotrypsin cleaves on the carboxy side of aromatic amino acids, so Y, W, and F would be cleaved and K would be untouched. K would be cleaved by trypsin.

In differential centrifugation, what is the correct order for fraction separation?

Nuclear, mitochondrial, membrane, cytosol Differential centrifugation uses increasing speed and time to separate cellular components. The nuclear fraction is the first fraction isolated, followed by the mitochondrial fraction. Next is the membrane fraction, which leaves the cytosol fraction containing soluble proteins.

The first step of protein mass spectrometry is to get the protein (usually peptide fragments) into a gas phase as an ion. Which of the following describes matrix-assisted laser desorption/ionization (MALDI) ionization?

Protein fragments are embedded in a solid mixture that absorbs light, and then a laser flashes on this mixture, leaving fragmented and ionized peptides in the gas phase.

Sodium dodecylsulfate (SDS) plays an important role in SDS PAGE. Select each correct description of what SDS does in denatured electrophoresis.

SDS is an amphipathic compound that binds to the hydrophobic portion of the protein, coating the mixture and giving the protein an overall negative charge proportional to the size of the protein. Because SDS is a detergent, it plays a role in denaturing the protein.

Using ion exchange and affinity chromatography, you have isolated a protein. To check on the size of the protein, you subject the purified protein to gel filtration chromatography and the protein elutes with a molecular mass of 140,000 Daltons. However, to show that the protein is purified, you perform SDS PAGE on the protein, and two bands with molecular masses of 25,000 and 45,000 Daltons are identified. Which answer best explains your results?

The native protein, as seen after gel filtration chromatography, has a molecular mass of 140,000 Daltons. But denatured protein, as seen during SD-PAGE, reveals that the protein is comprised of four subunits: two 25,000-Dalton subunits and two subunits at 45,000 Daltons.

Using immunoprecipitation, you can isolate a protein (protein X) you think is involved in chronic myelogenous leukemia (CML), which is caused, in part, by a hyperactive tyrosine protein kinase called ABL. You think one of the targets of ABL is the protein X that you can purify. In comparing tissues with and without the disease, you subject the samples to isoelectic focusing. Understanding how IEF works, how would you expect the samples with CML to migrate compared to the wild-type, non-diseased sample protein? Use the figure to help you consider your response.

The phosphorylated protein will have a lower isoelectric point and thus migrate further toward the anode.

Which of the following statements best describes the difference between a primary and secondary antibody as used in a Western blot?

The primary antibody binds the antigen, whereas the secondary antibody binds the primary antibody and serves to detect the complex with its linked enzyme.

Assemble the peptides, review the sites of proteolysis, and determine the full sequence of the peptide produced by this bacteria. ChymotrypsinW AGHIES GVCAQIT HWQGKGAGESF HGESDVIQRTSEGF TrypsinWHWQGK TSEGFAGHIES GAGESFGVCAQITHGESDVIQR

WHWQGKGAGESFGVCAQITHGESDVIQRTSEGFAGHIES

Which of the following is incorrect concerning protein structure determination by X-ray crystallography? X-ray crystallography provides dynamic information about protein structure. You Answered Highly ordered crystals are required. Isomorphous replacement is used to aid phase determination. A 1.5 Å resolution structure is better than a 3 Åresolution structure.

X-ray crystallography provides dynamic information about protein structure. X-ray crystallography requires high quality, highly ordered crystals to provide a diffraction pattern that can be interpreted. This means that the proteins in the crystal cannot have multiple conformations, so X-ray crystallography provides a "snapshot" of the protein in a particular state and not dynamic information. Determining the phase can be challenging, but the use of heavy atom derivatives, which are electron dense, helps and is called isomorphous replacement. The smaller the angstrom value, the higher the resolution of a structure.

To specifically isolate a negatively charged protein, _____________ chromatography would be most useful.

anion-exchange Gel filtration chromatography is used for separating proteins based on size. Affinity chromatography uses the specific interaction between a protein and an immobilized ligand to isolate a protein of interest. Ion exchange chromatography separates proteins based on charge differences. Anion-exchange resins bind negatively charged proteins, while cation-exchange resins bind positively charged proteins.

Secondary antibodies have the following properties EXCEPT that they are typically linked to an enzyme that generates a colorimetric or fluorescent product. recognize a primary antibody as its antigen. are typically polyclonal. are typically from the same animal as the primary antibody.

are typically from the same animal as the primary antibody. Secondary antibodies are polyclonal antibodies that are able to recognize the primary antibody from a different animal. For example, a secondary antibody from a goat can be used to bind to a primary antibody from a rabbit. Secondary antibodies are typically linked to enzymes to produce a measurable signal for location determination on the blot.

Extracting a protein bound to a column is called which of the following?

elution

Polyclonal antibodies differ from monoclonal antibodies in that polyclonal antibodies are a __________ mixture of immunoglobulin proteins that recognize __________ on an antigenic protein.

heterogeneous; one or more epitopes Polyclonal antibodies are a heterogeneous mixture of immunoglobulin proteins that recognize one or more epitopes on an antigenic protein. Monoclonal antibodies are a homogeneous mixture of immunoglobulin proteins that recognize a single epitope on an antigenic protein. Monoclonal antibodies are more specific, but are also more challenging to generate.

Which of the following is one of the critical concerns when purifying a protein?

identifying the protein, often by a biochemical assay

The purpose of using two different protein-specific monoclonal antibodies in ELISA is to

improve specificity by forming a "sandwich" with the antigen protein in the middle. Two different monoclonal antibodies are used in ELISA to form a sandwich. Each antibody binds to a different epitope, which will improve specificity. One of the antibodies is immobilized and serves to capture the protein of interest, while the second antibody detects the bound protein. This detection antibody is recognized by the signaling antibody, so the sandwich format is necessary to produce a signal in the corresponding assay plate well.

Generally, the chemistry of Fmoc blocking is straightforward for most amino acids during solid state peptide synthesis. There is one amino acid, however, that presents a problem for Fmoc blocking during solid state peptide synthesis. That amino acid is

lysine

In an experiment, you found two proteins (protein X and protein Z) with similar size and sequence homology. Two domains of both proteins are nearly identical, but each possesses a different sequence in another domain. Which antibody would be the best selection for you to identify the expression of protein Z but not protein X using a Western blot?

monoclonal antibodies generated against the unique domain

The specificity of monoclonal antibodies is determined

with screening of hybridomas. Each B cell makes one type of antibody, so the B cells need to be separated out to identify antigen-specific antibodies. This separation is done by forming a hybridoma, which is when a B cell is fused with an immortalized tumor cell. These hybridomas can be cultured and screened for antibody production. This screening process separates out the desired antibody-producing hybridomas, which can then be cloned and cultured to produce monoclonal antibodies.