Biochem chapter 5

You are employed in a prominent biochemistry lab, ProteinsRUs. The company offers a $500 bonus to the person who can develop a way to perform Western blotting without having to wait to make a unique antibody for proteins under analysis. How would you propose to do this? Explain the method you would use in two to three sentences and give an example.

A faster way to perform the analysis would be to use commercially available epitopetags and gene-encoded proteins. The FLAG and myc epitopes sequences are highly antigenic, and their coding sequences can easily be added to the protein coding sequences of genes using conventional recombinant DNA techniques. This would eliminate the need to wait for antibodies to be isolated from animals.

Describe how polyclonal antibodies are generated.

Either a rabbit, chicken, or goat is injected several times over the course of 6 to 8 weeks with an antigen. An immune response leads to the production of B-cells secreting antigen-specific antibodies. Whole blood is removed from the animal and the red blood cells are separated from the sample. The antigen-specific polyclonal antibodies are purified from the remaining animal serum using affinity chromatography.

How does the directionality of synthesis differ with in vivo polypeptide synthesis compared to solid-phase peptide synthesis?

In in vivo polypeptide synthesis, amino acids are added one at a time to the carboxyl terminus of the polypeptide chain. In solid-phase peptide synthesis, the amino acids are added to the amine terminus.

Which technique ionizes polypeptides by embedding the tryptic fragments into a light-absorbing matrix and exposing it to a laser? ESI MALDI NMR X-ray crystallography

MALDI

Why is X-ray crystallography more commonly used than NMR spectroscopy when determining protein structure?

NMR is limited to proteins that are less than ~100 kDa. This occurs because large molecules reorient slowly in solution, resulting in signals that average out and are lost during the process. Additionally, NMR requires more protein sample than X-ray crystallography

Describe the major difference between the types of protein samples used in X-ray crystallography and NMR spectroscopy. Then explain how this difference limits each method.

Protein samples used in NMR are in solutions, whereas the protein samples used in X-ray crystallography are in crystal form. In X-ray crystallography, it is sometimes hard to obtain crystals of a new protein because the conditions needed to precipitate a crystal are unknown. NMR spectroscopy requires a high protein concentration, which can be hard to obtain

Explain how proteins are separated using isoelectric focusing.

Proteins are applied to a gel strip containing a stable pH gradient. An electric field is applied and the proteins migrate toward the anode (if they are cations) or the cathode (if they are anions). Once the protein reaches its isoelectric point, where the charge is zero, it will stop migrating.

Explain the difference between the primary and secondary antibody using a Western blot.

The primary antibody facilitates the antigen-antibody interactions. The secondary antibody is covalently linked to an enzyme that catalyzes a chemical reaction that can be detected by a colorimetric or fluorometric assay when the substrate is added.

Explain how protein samples that contain the antigenic protein are recognized using a Western blot.

The proteins are separated using gel electrophoresis and transferred to a nitrocellulose membrane. The membrane is treated with a blocking solution to prevent nonspecific antibody binding, and then it is incubated with the primary antibody, which facilitates antigen-antibody binding. The primary antibody is removed with washing and the membrane is incubated with a secondary antibody. The secondary antibody is linked to an enzyme that causes a chemical reaction that can be detected using either colorimetric or fluorometric assays.

Explain how to separate the following proteins using two different types of chromatography Proteins. pI. Molar mass 1. 3.8. 75 2. 7.5. 118 3. 4.0. 120

Use size exclusion chromatography to separate protein 1 from 2 and 3. Then use ion-exchange chromatography to separate 2 and 3. Because proteins 2 and 3 have such different pIs, using a buffer with a pH of 4.0 would facilitate the separation. Protein 3 would effectively have no charge, whereas protein 2 would be charged at that pH. The charge difference would result in different elution rates of the protein.

In solid-phase peptide synthesis, which reagent is used to remove the peptide from the solid resin support? a. HF b. DCC c. Fmoc d. PITC

a. HF

In isoelectric focusing, a protein with a pH below the pI would a. migrate toward the anode. b. migrate toward the cathode. c. stop migrating. d. migrate, but there is no way to determine the direction.

a. migrate toward the anode

The advantage of using an SDS-PAGE gel compared with a native PAGE gel is that the SDS-PAGE gel a. separates proteins only based on molar mass. b. gives information about the charge or conformation of the protein. c. results in a better separation of small proteins. d. increases the resolution of large and small proteins.

a. separates proteins only based on molar mass.

During solid-phase peptide synthesis, dicyclohexylcarbodiimide is used a. as a blocking agent. b. as an activating agent. c. to cleave the peptide from the resin. d. to couple amino acids during the synthesis

b. as an activating agent.

In tandem mass spectrometry, the second mass spectrometer a. determines the nuclear spin of the atoms. b. determines the mass of peptide subfragments. c. uses bioinformatics to compare the masses of the peptides. d. uses electrospray ionization to select peptide fragments.

b. determines the mass of peptide subfragments.

One of the most difficult steps in X-ray crystallography is a. using the correct radio frequency pulses to perturb the nuclear spin. b. determining the phases of diffracted X-rays. c. dissolving the protein in the appropriate solvent. d. obtaining a large enough sample for analysis.

b. determining the phases of diffracted X-rays.

The advantage of using a native PAGE gel compared with an SDS-PAGE gel is that the native PAGE gel a. separates proteins only based on molar mass. b. gives information on the charge or conformation of the protein. c. results in a better separation of small proteins. d. increases the resolution of large and small proteins.

b. gives information on the charge or conformation of the protein.

Which peptide would migrate the slowest in an SDS-PAGE gel? a b c d

c

When preparing to isolate proteins from plant cells, the first step in preparing the cell homogenate would be a. sonication. b. using a French press. c. treatment with mild detergents. d. enzymatic treatment

d. enzymatic treatment

The figure below shows how differential centrifugation can be used to isolate four subcellular fractions. Which fraction corresponds to the mitochondrial fraction? 1 2 3

2

The figure below shows how differential centrifugation can be used to isolate four subcellular fractions. Which fraction corresponds to the cytosol fraction? 1 2 3 4 5 6

3

The figure below shows how differential centrifugation can be used to isolate four subcellular fractions. Which fraction corresponds to the membrane fraction? 1 2 3

3

Below are the steps of solid-phase peptide synthesis: 1. The protecting groups of the amino acids are removed. 2. The peptide is released from the solid support resin. 3. An incoming amino acid is activated at the carboxyl group by DCC and added to the column. 4. Fmoc is removed by treatment with a base and the C-terminal amino acid is attached to the resin molecule. 5. The resin bound C-terminal amino acid and the incoming N-terminal amino acid are coupled. What is the appropriate order of these steps?

4, 3, 5, 1, 2

Below are the steps of a Western blot. What is the correct order? 1. The membrane is incubated with the secondary antibody. 2. The membrane is incubated with the primary antibody. 3. The membrane is washed with a blocking solution. 4. Proteins are transferred to a nitrocellulose membrane. 5. Protein extracts are separated by gel electrophoresis.

5,4,3,2,1

Explain the difference between polyclonal and monoclonal antibodies.

A polyclonal antibody is a heterogeneous mixture of immunoglobulin proteins and a monoclonal antibody is a homogenous mixture of immunoglobulin proteins. A polyclonal antibody recognizes one or more epitopes, whereas a monoclonal antibody only recognizes a single epitope

Compare and contrast the three MOST commonly used homogenization techniques to prepare a cell extract.

All three methods are used to disrupt or remove the cell membrane to prepare the cell homogenate. Sonication uses ultrasonic waves to disrupt the membrane through vibrational effects. Shearing uses mechanical force by pushing the cells through a small opening. Cells can also be incubated with detergents to disrupt the membrane.

Compare and contrast electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI).

Both methods are used to ionize peptides without disintegrating them. ESI releases tryptic fragments (polypeptides) from a small metallic capillary at high voltage, causing the peptides to evaporate, forming a highly charged molecule in the gas phase. In MALDI the cleavage fragments are embedded in a light-absorbing matrix and released as a charged molecule after the flash of the laser.

Theoretically, an individual B cell could express any combination of light chain and heavy chain polypeptides to produce one of 10 antibody complexes. How does this seemingly limited production of antibodies protect us from the tens of millions of foreign antigens we encounter through our lives?

DNA recombination is the reason that we produce a multitude of unique antibodies to protect us from the tens of millions of foreign antigens. Variable domain coding sequences are joined in small DNA segments on the same chromosome. These random DNA recombination events are what generate the unique antibodies.

Describe how an ELISA detects antigenic proteins.

ELISA uses a "sandwich" method where a primary monoclonal antibody is covalently attached to the bottom of a 96-well plate. This antibody recognizes a single epitope on the antigen. The sample is added to the well plate and incubated for several hours while the antigen-antibody binding takes place. A second primary monoclonal antibody is added to the well, recognizing a distinct epitope on the antigenic protein. A third antibody is added that recognizes the detection antibody and causes a chemical reaction that can be detected using either colorimetric or fluorometric assays.

Which technique ionizes polypeptides by releasing them from a small metallic capillary at high voltage? ESI MALDI NMR X-ray crystallography

ESI

What is the advantage of using sodium dodecyl sulfate (SDS) in gel electrophoresis?

Gel electrophoresis separates molecules based on size and charge. SDS is an amphipathic molecule that masks the charges on the protein. Additionally, the SDS denatures the protein, negating the effects of shape on migration. This means that an SDS-PAGE gel separates the proteins as an approximate function of molar mass.

Explain the differences among the three types of column chromatography.

Gel filtration chromatography (size exclusion chromatography) separates proteins based on their size using carbohydrate beads that retain proteins in pores. Ion-exchange chromatography contains either cation or anion resins that separate proteins based on size. Anion exchange binds to negatively charged proteins, whereas cation exchange binds to positively charged proteins. Affinity chromatography separates proteins using specific binding properties.

What are the advantages of using a monoclonal antibody compared with a polyclonal antibody?

Monoclonal antibodies are generated from an immortalized hybridoma cell line, resulting in an unlimited supply of antibodies. The polyclonal antibody supply is limited because the animal eventually dies. Additionally, monoclonal antibodies provide the ability to monitor quality continually using standardized diagnostic measures.

Analyze and interpret the 2-D PAGE gel shown below. What information can be determined about the proteins labeled A and B? A is higher and more to the left of B

Protein A has a higher mass and lower pI than protein B. Protein B will have a higher pI and a lower

Contrast the way that SDS-PAGE and native PAGE gels separate proteins.

SDS-PAGE gels use several chemicals (including the SDS) that denature the protein into polypeptide chains. The addition of the SDS negates the charge and the shape of the protein, so the protein is only separated based on molar mass. In a native PAGE, the protein is not denatured and is separated based on size, molar mass, and charge.

Why is it more advantageous to use Edman degradation instead of Sanger's sequencing method?

Sanger's method requires adding fresh protein after each cleavage, making the process labor intensive. The method also requires a relatively large amount of protein. The Edman degradation does not require fresh protein to be added after each step, so a much smaller amount of protein is needed.

Compare the order of migration and the separation of three proteins (25 kDa, 100 kDa, and 150 kDa) in an SDS-PAGE gel with 15% polyacrylamide gel.

The 25 kDa protein would migrate the farthest, followed by the 100 kDa and 150 kDa protein. In SDS-PAGE gels, the smallest proteins migrate faster. There would be good separation between the 25 kDa protein and the other two proteins. However, there would be more resolution between the 100 kDa and the 150 kDa proteins because of the high-percentage polyacrylamide gel.

How can the mass of an unknown protein can be determined using gel electrophoresis?

The mass of an unknown protein can be determined by comparing the migration distance in the gel to that of known proteins that serve as the molecular mass markers. The log of the masses of the known proteins can be plotted against the migration distance, forming a straight line. The molecular weight of the unknown protein can then be determined based on its migration distance in the same gel.

Why is protein purification often referred to as an art and a science?

There are between 10,000 and 100,000 different proteins in a cellular sample. To isolate just one requires that scientists rely on the differences in the chemical and physical properties of the individual proteins. Not all processes are going to work for the same protein, so a scientist must develop new protocols for each one. A scientist must rely on both the quantitative and qualitative properties of the protein to do this.

A polypeptide was digested by trypsin and chymotrypsin. Use the following information to determine the polypeptide sequence. Trypsin Chymotrypsin YMR MR LSSWEMYEK VEF VEFDGSK EMY GAWAK DGKLSSW EKGAW AKY

VEFDGSKLSSWEMYEKGAWAKYMR

How does NMR spectroscopy differ from X-ray crystallography when determining the structure of a protein?

X-ray crystallography gives the atomic positions of the protein in a static crystal lattice. NMR spectroscopy uses a high-strength magnetic field to determine the relative position of specific atoms in a protein solution.

Which of the following peptides would be eluted last when separated using gel filtration chromatography? a b c d

a

Which peptide would migrate the fastest in a SDS-PAGE gel? a b c d

a

Which percentage of polyacrylamide would give the best separation for large proteins? a. 5% b. 7.5% c. 10% d. 20%

a. 5%

What is the advantage of using polyclonal antibodies compared with monoclonal antibodies? a. They are less labor intensive to generate. b. They provide the ability to monitor quality continually. c. The process used to produce the antibodies provides unlimited supplies. d. They can be generated without using animals.

a. They are less labor intensive to generate

Which type of chromatography separates proteins using specific binding properties? a. affinity chromatography b. gel filtration chromatography c. size exclusion chromatography d. high-performance liquid chromatography

a. affinity chromatography

During solid-phase peptide synthesis, 9-fluorenylmethoxycarbonyl is used a. as a blocking agent. b. as an activating agent. c. to cleave the peptide from the resin. d. to couple amino acids during the synthesis.

a. as a blocking agent.

The specific sites on the antigen that interacts with the antibody are called a. epitopes. b. immunoglobin light chains. c. immunoglobin heavy chains. d. variable-domain amino acid residues.

a. epitopes

Protein-specific antibodies can be used to detect proteins within cells conserving the cell architecture using a. immunofluorescence. b. Western blot. c. immunoprecipitation. d. ELISA.

a. immunofluorescence.

In isoelectric focusing a. negatively charged proteins migrate toward the cathode. b. negatively charged proteins migrate toward the anode. c. small proteins migrate the fastest. d. large proteins migrate the fastest.

a. negatively charged proteins migrate toward the cathode.

Monoclonal antibodies a. recognize a single epitope. b. recognize multiple epitopes. c. are isolated from leukocytes. d. are isolated from red blood cells

a. recognize a single epitope.

Biochemical assays are important in the process of isolating proteins because they a. uniquely identify one protein. b. separate proteins based on polarity. c. isolate proteins based on size. d. determine the molecular weight of proteins.

a. uniquely identify one protein.

The majority of serum proteins and nonspecific antibodies are removed during the generation of polyclonal antibodies by a. washing with a low pH buffer. b. gel electrophoresis. c. size exclusion chromatography. d. salting out.

a. washing with a low pH buffer.

Predict the fragments of the following peptide after cleavage by trypsin. GLMKTYPDSTA a. GLM KTYPDSTA b. GLMK TYPDSTA c. GLMKTY PDSTA d. GLMKTYPD STA

b. GLMK TYPDSTA

Co-immunoprecipitation proteins are separated through a. affinity chromatography. b. SDS-PAGE. c. HPLC. d. size exclusion chromatography.

b. SDS-PAGE.

Most secondary antibodies used in Western blots are covalently linked to the enzyme a. luciferase. b. horseradish peroxidase. c. catalase. d. hydrolase.

b. horseradish peroxidase.

Epitopes on protein antigens are generally located a. in the heavy chain domains. b. on polar surface groups in loop regions. c. in the light chain domains. d. in association with variable domain amino acid residues.

b. on polar surface groups in loop regions.

In isoelectric focusing a. positively charged proteins migrate toward the cathode. b. positively charged proteins migrate toward the anode. c. small proteins migrate the fastest. d. large proteins migrate the fastest.

b. positively charged proteins migrate toward the anode.

The part of the Western blot that contains the protein-specific recognition and facilitates the antigen-antibody interactions is the a. SDS-PAGE gel. b. primary antibody. c. tertiary antibody. d. filter membrane.

b. primary antibody.

Which separation technique exploits the solubility differences of proteins? a. centrifugation b. salting out c. dialysis d. ion-exchange chromatography

b. salting out

Calculate the purification of the target protein when there is a 30% decrease in activity and a 55% decrease in total protein after centrifugation. a. 0.55-fold b. 0.64-fold c. 1.6-fold d. 1.8-fold

c. 1.6-fold

After centrifugation, there is a 10% decrease in activity and a 75% decrease in total protein. What is purification of the target protein? a. 0.28-fold b. 1.3-fold c. 3.6-fold d. 7.5-fold

c. 3.6-fold

Calculate the specific activity when 500 mg of protein has an activity of 18,000 units. a. 0.28 units/mg protein b. 36 units/mg protein c. 9000 units/mg protein d. 13,000 units/mg protein

c. 9000 units/mg protein

A polypeptide was digested by trypsin and chymotrypsin. Use the following information to determine the polypeptide sequence. a. ECFGMREDWKYLM b. LMYKWMGFCEDER c. LMYKWDERMGFCE d. CELMYKWDERMGF

c. LMYKWDERMGFCE

Why is the supply of polyclonal antibodies limited? a. They are time consuming to produce. b. The yield is very low. c. The animals the antibodies are isolated from eventually die. d. The production is limited by the government.

c. The animals the antibodies are isolated from eventually die.

What is the advantage of using monoclonal antibodies compared with polyclonal antibodies? a. They are less labor intensive to generate. b. They can recognize more than one epitope. c. The process used to produce the antibodies provides unlimited supplies. d. They can be generated without using animals.

c. The process used to produce the antibodies provides unlimited supplies.

Polyclonal antibodies are a. derived from B cells isolated from the spleen. b. isolated from hybridoma cells. c. a heterogeneous mixture. d. a homogenous mixture.

c. a heterogeneous mixture.

The Fab immunoglobin fragment a. is generally found within β -barrels. b. is located in polar surface groups in loop regions. c. contains the high-affinity antigen binding site. d. is responsible for the solubility of the antibody

c. contains the high-affinity antigen binding site.

During a Western blot, the membrane is washed with a blocking solution before being treated with the primary antibody to a. retain the position of the proteins in the transfer from gel to membrane. b. facilitate antibody-antigen interactions. c. decrease nonspecific antibody binding. d. facilitate a chemical reaction that can be detected

c. decrease nonspecific antibody binding

After using the salting out method to isolate proteins, how is ammonium sulfate removed from the solution? a. sonication b. French press c. dialysis d. centrifugation

c. dialysis

When cells isolated from the spleen are fused with long-living cells, they produce a. B cells. b. immortalized tumor cells. c. hybridoma cells. d. red blood cells.

c. hybridoma cells

Which technique can be combined with mass spectrometry to identify protein antigens in large cellular complexes? a. immunofluorescence b. Western blot c. immunoprecipitation d. ELISA

c. immunoprecipitation

Protein NMR is more useful than X-ray crystallography for studying a. secondary structure elements. b. large proteins. c. protein unfolding. d. static protein structures.

c. protein unfolding.

Which component used in a Western blot is covalently linked to an enzyme? a. SDS-PAGE gel b. primary antibody c. secondary antibody d. filter membrane

c. secondary antibody

In monoclonal antibody generation, the cells that produce the antibody in the animal are located in __________ cells. a. heart b. liver c. spleen d. red blood

c. spleen

Which method uses mass to charge ratios as well as a bioinformatics approach to determine the amino acid composition of a protein? a. X-ray crystallography b. nuclear magnetic resonance c. tandem mass spectrometry d. size exclusion chromatography

c. tandem mass spectrometry

The following peptides are separated using an anion exchange resin in ion-exchange chromatography. Which peptide is eluted first? a b c d

d

Which percentage of polyacrylamide would give the best separation for small proteins? a. 5% b. 7.5% c. 10% d. 20%

d. 20%

Predict the fragments of the following peptide after cleavage by the V-8 protease. CMVAADGKLEPVS a. CMVAAD GKLEPVS b. CMV AADGKLE PVS c. CMVAADG KLEPVS d. CMV AAD GKLE PVS

d. CMV AAD GKLE PVS

Which technique uses highly specific monoclonal antibodies in a high-throughput form to detect small amounts of antigen? a. immunofluorescence b. Western blot c. SDS-PAGE gel d. ELISA

d. ELISA

Why is the process of purifying proteins from cells considered challenging? a. Proteins are insoluble. b. The process is expensive and time consuming. c. There is no way to determine the structure of the protein. d. There are 10,000 to 100,000 proteins in one sample.

d. There are 10,000 to 100,000 proteins in one sample.

During the production of polyclonal antibodies, how are the antigen-specific antibodies purified? a. dialysis b. gel electrophoresis c. size exclusion chromatography d. affinity chromatography

d. affinity chromatography

During the production of polyclonal antibodies, where are the antibodies isolated from? a. an immortalized tumor cell line b. liver cells c. spleen cells d. blood cells

d. blood cells

Which enzyme or reagent cleaves a peptide at the carboxyl side of a methionine residue? a. trypsin b. chymotrypsin c. V-8 protease d. cyanogen bromide

d. cyanogen bromide

The size of proteins studied by NMR is limited because a. a small sample size is needed. b. it is hard to determine the phases of diffracted X-rays. c. the measurements of the distances between atoms are only approximate. d. large molecules reorient slowly in solution.

d. large molecules reorient slowly in solution

Which animal is most often used to generate monoclonal antibodies? a. rabbit b. chicken c. goat d. mouse

d. mouse

Two-dimensional polyacrylamide gel electrophoresis separates proteins based on a. pI and shape. b. ligand affinity and molecular weight. c. shape and ligand affinity. d. pI and molecular weight.

d. pI and molecular weight

If a protocol for a Western blot calls for the use of rabbit anti-mouse antibody, what type of antibody would this be considered? a. monoclonal primary b. polyclonal primary c. monoclonal secondary d. polyclonal secondary

d. polyclonal secondary

Immunoprecipitation uses an antibody-bead combination to a. detect the antigen. b. enrich the protein solution. c. digest the proteins for use in mass spectrometry. d. separate the proteins using centrifugation.

d. separate the proteins using centrifugation.

After centrifugation, the purity of the protein is determined by a. absorbance measurements. b. activity units. c. total protein content. d. specific activity.

d. specific activity.

In tandem mass spectrometry, the first mass spectrometer a. determines the nuclear spin of the atoms. b. determines the mass of peptide subfragments. c. uses bioinformatics to compare the masses of the peptides. d. uses electrospray ionization to select peptide fragments

d. uses electrospray ionization to select peptide fragments

The specific sites on the antibody that interacts with the antigen are a. epitopes. b. immunoglobin light chains. c. immunoglobin heavy chains. d. variable-domain amino acid residues.

d. variable-domain amino acid residues.

In an ELISA, the capture antibody is a __________ antibody

monoclonal primary

In an ELISA, the detection antibody is a __________ antibody monoclonal primary polyclonal tertiary monoclonal secondary polyclonal secondary

monoclonal primary

The figure below shows three proteins that are separated using gel filtration chromatography. Which protein is the largest? squares circles triangles Not enough information is included to determine the protein.

squares