LS 23L Lab F - Gel Electrophoresis

SDS can break covalent bonds that hold the protein subunits together True or False

False

Spectrophotometry provides a ______ measure of the concentration of DNA. Qualitative or Quantitative?

Quanititative

T/F When absorption is >1.8, it indicates good nucleic acid purity

T

According to the lab safety sheet, the agarose gels used in this lab are cast with GelGreen, a less toxic alternative to ethidium bromide. True or False

True

T/F The agarose gels used in this lab are cast with GelGreen, a less toxic alternative to ethidium bromide

True

Protein X is a protein that is composed of four subunits of equal size--alpha, beta, delta, and gamma--that are bound by either noncovalent interactions or disulfide bridges. Only the beta and delta subunits are bound to each other by disulfide bridges. How many bands would you expect to see in an SDS PAGE experiment in which beta-mercaptoethanol, a reducing agent, is used? a. 1 b. 4 c. None d. 2 e. 3

a. 1

You find that the molecular weight of your native protein is 200kD. If your protein was composed of only one type of subunit,which of the following sizes can be correct for the subunits of the native protein? a. 25kD. b. 150kD. c. 7kD. d. 51kD. e. 3kD.

a. 25kD.

How does the method of gel filtration differ from that of SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)? Select one: a. Gel filtration provides an estimate of the molecular weight of a protein in its native, intact form, while SDS-PAGE denatures proteins by disrupting noncovalent linkages between the subunits. b. Gel filtration breaks noncovalent linkages between protein subunits, while SDS-PAGE breaks both noncovalent linkages and disulfide bonds between subunits. c. Gel filtration keeps the protein in its native, intact form, while SDS-PAGE uses an electrical current to break disulfide bonds between protein subunits. d. Both gel filtration and SDS-PAGE break disulfide bonds between protein subunits, but SDS-PAGE also denatures proteins by breaking noncovalent linkages between subunits. e. Gel filtration breaks disulfide bonds so that proteins separate into individual subunits, while SDS-PAGE keeps disulfide bonds intact and allows visualization of the entire protein using electrophoresis.

a. Gel filtration provides an estimate of the molecular weight of a protein in its native, intact form, while SDS-PAGE denatures proteins by disrupting noncovalent linkages between the subunits.

A student runs an experiment using Gel filtration and SDS-PAGE to find the molecular composition of his protein. The weight obtained from gel filtration was 400 daltons. When he ran the SDS-PAGE with the reducing agent, he got 3 different lines: one A at 25 daltons, B at 50 daltons and C at 75 daltons. What could be the composition? I. 2 of A, 4, of B, and 2 of C II. 4 of A and 4 of C III. 9 of A, 2 of B, and 1 of C Select one: a. I or III b. II only c. I only d. II or III e. I or II or III

a. I or III

Which of the following statements is TRUE concerning the detergent SDS? a. It coats proteins with a negative charge, with equal amount of charge per unit length. b. For every two amino acids, an average of four SDS molecules associate with them. c. It helps separate protein subunits by their charges. d. Smaller protein subunits associate with more SDS molecules than do larger subunits. e. In a multisubunit polypeptide, it breaks apart disulfide linkages.

a. It coats proteins with a negative charge, with equal amount of charge per unit length.

Which of the following best describes the function of polyacrylamide gel used in SDS-PAGE? a. It separates proteins based on size due to the web-like matrix formed by its molecules. b. It acts as the reducing agent which breaks down disulfide bonds within the protein. c. It contains enzymes that break down complex proteins into its major subunits. d. It binds to the protein bands and allows them to be seen under UV light e. It is very positively charged so that negatively charged proteins tightly adhere to it.

a. It separates proteins based on size due to the web-like matrix formed by its molecules.

Characterize the difference between the interaction that the detergent sodium dodecyl sulfate (SDS) has with proteins and the interaction that reducing agents such as b-mercaptoethanol have with proteins. a. SDS is anionic and binds to every side chain of proteins, disrupting ionic and hydrogen bonds, while reducing agents such as b-mercaptoethanol disrupt covalent bonds b. SDS is nonionic and at low concentrations is binds to every side chain of proteins, disrupting ionic and hydrogen bonds, while reducing agents such as b-mercaptoethanol disrupt covalent bonds. c. SDS is a detergent with neutral overall charge, which forms micelles with the proteins. d. SDS is cationic and at high concentrations it binds to every side chain of a protein, disrupting covalent bonds, while reducing agents such as b-mercaptoethanol disrupt ionic and hydrogen bonds.

a. SDS is anionic and binds to every side chain of proteins, disrupting ionic and hydrogen bonds, while reducing agents such as b-mercaptoethanol disrupt covalent bonds

You want to determine the distribution of subunits in a given protein. Through gel filtration you determine that the protein is 140 kDs. SDS-PAGE analysis gives three bands that correspond to 12, 25, and 30 kDs. Which of the following is a possible explanation for this result? Select one: a. The protein contains five 12 kD subunits, two 25 kD subunits, and one 30 kD subunit. b. The protein contains two subunits of each size. c. There is not enough information given to determine the number of subunits in this protein. d. The protein contains nine 12 kD subunits, one 25 kD subunit, and one 30 kD subunit. e. The proteins contains two 25 kD subunits and three 30 kD subunits.

a. The protein contains five 12 kD subunits, two 25 kD subunits, and one 30 kD subunit.

Which of the following techniques allows the determination of a protein's mass in its native form? Select one: a. gel filtration b. SDS-page c. Western Blot d. RASMOL e. ultracentrifugation

a. gel filtration

Which of these substances is usually used to visualize proteins on a POLYACRYLAMIDE GEL? a) aniline blue b) coomassie blue c) phenol red d) ethidium bromide e) alcian blue

b) coomassie blue wrong look this up in the manual

When biologists try to separate proteins on the basis of size, they will often use an SDS-PAGE gel. Which of the following is true about the materials used in this process? Select one: a. A marker contains proteins of unknown molecular weights and is compared against proteins of known molecular weight. b. A reducing agent is used to disrupt disulfide bonds. c. SDS is a cationic detergent that denatures proteins. d. SDS is a detergent used to disrupt covalent bonds. e. An agarose gel allows ionized proteins to filter according to relative size.

b. A reducing agent is used to disrupt disulfide bonds.

What are the functions of SDS in gel electrophoresis for estimating protein sizes? I. Disrupts hydrogen bonding in proteins, linearizing the protein II. Provides an overall negative charge on proteins, making the migration distance on gel a function of only protein size III. Intercalates between the amino acids of the protein allowing it to be visualized on the gel a. III only b. I and II c. II only d. I only e. I, II, and III

b. I and II

During the SDS-PAGE lab, the TA forgot to add a loading buffer with the protein sample, which usually contains a reducing agent and SDS. What would you expect to happen from this? Select one: a. The experiment would run normally even without the loading buffer. b. Neutral proteins would remain in the well. Negatively charged proteins would migrate slightly into the gel. Positively charged proteins would migrate out of the well. c. The experiment would run slower than usual without the loading buffer. d. If the protein does not contain any disulfide bond, the experiment will run as usual. However, if the protein contains disulfide bond(s), the subunits will not be separated. e. Proteins with smaller mass will migrate further than those with a larger mass because of the even mass-to-charge ratio.

b. Neutral proteins would remain in the well. Negatively charged proteins would migrate slightly into the gel. Positively charged proteins would migrate out of the well.

What kind of gel medium is used in SDS-PAGE? a. TBE b. Polyacrylamide c. FITC d. Agarose e. Ethidium Bromide

b. Polyacrylamide

What is the difference between SDS's function and reducing agent's function in SDS-PAGE? Select one: a. SDS causes the protein subunit to migrate in the gel but reducing agent just breaks the bonds between subunits b. SDS breaks disulfide bonds and reducing agent breaks the non covalent bonds c. They both act in the same way and there is no difference between them d. SDS breaks the non covalent bonds and reducing agent breaks the disulfide bonds e. There is no reducing agent used in the SDS-PAGE and only SDS is used

b. SDS breaks disulfide bonds and reducing agent breaks the non covalent bonds

Ricky used SDS-PAGE to find the molecular weight of Protein X's subunits. After running the gel, he observed two bands, one at 30kD and another at 40kD. If Ricky knows the molecular weight of the native protein is 210kD, which of the following is/are the best conclusion(s)? Select one: a. None of these b. There must be three 30kD subunits and three 40kD subunits. c. All of these d. Protein X is a homodimer. e. Protein X is a heterodimer.

b. There must be three 30kD subunits and three 40kD subunits.

After the treatment of proteins described in the lab manual, all proteins used in this lab should carry an overall ______ charge. Select one: a. positive b. negative

b. negative

The combination of gel filtration and SDS-PAGE can reveal the ______ structure of a protein. a. tertiary b. quaternary c. secondary d. primary

b. quaternary

The combination of gel filtration and SDS-PAGE can reveal the ______ structure of a protein. Select one: a. secondary b. quaternary c. primary d. tertiary

b. quaternary

Which of the following is not a similarity between performing a gel electrophoresis analysis on protein versus DNA? a. Both methods utilize electrical currents to mobilize the macromolecules. b. If performed correctly, the samples always move towards the anode in both methods. c. Both methods utilize SDS to create a uniform charge to mass ratio. d. Agarose gels would work for both protein and DNA gel electrophoresis. e. The larger the macromolecules are, the slower they migrate through the gel matrix in both methods.

c. Both methods utilize SDS to create a uniform charge to mass ratio.

What is the name of the method for separating charged molecules, such as proteins and nucleic acid, in an electrical field? Select one: a. Electroseparation b. Electroporation c. Electrophoresis d. Chemophoresis e. Denaturation

c. Electrophoresis

You want to analyze the concentration of a solution. You take your solution to a spectrometer and record the absorbance reading. Which statement is correct regarding spectrometer function? a. Light passes through the sample. Some light is absorbed and some passes through. The spectrometer gives a value for the amount of light which passes through. b. Electrons are passed through the sample. The spectrometer gives a value for the amount of light particles excited by the absorbed electrons. c. Light passes through the sample. The spectrometer yields a value for the amount of light absorbed by the solution. d. Running a solution through a spectrometer is not a practical method of measuring absorbance. e. Electrons are passed through the sample. The spectrometer reads a value for the fraction of electrons absorbed by the solution.

c. Light passes through the sample. The spectrometer yields a value for the amount of light absorbed by the solution.

Which of the following is a difference between doing SDS-PAGE and using Agarose gel? a. Only protein mass can be estimated by Agarose b. Only protein mass can be estimated by SDS c. SDS-PAGE tends to be more accurate. d. Only Agarose can be done vertically e. Only SDS can be done vertically

c. SDS-PAGE tends to be more accurate.

Some of the chemicals/materials you will use in the Polyacrylamide and Agarose Gels lab include (check all that apply): Select one or more: a. Ethidium bromide b. Chloroform c. TGS (tris-glycine-SDS) buffer d. none of these e. Tris glycine polyacrylamide gels

c. TGS (tris-glycine-SDS) buffer e. Tris glycine polyacrylamide gels

Agarose gel electrophoresis as used in the lab you will be performing this week can do all of the following except: Select one: a. show whether the DNA is the correct size b. confirm the presence of purified DNA visually c. give a sequence for the amplified DNA d. show whether the DNA product is composed primarily of primer-dimer e. give a qualitative overview of the DNA purity

c. give a sequence for the amplified DNA

There are two methods for estimating the size of proteins: gel filtration and SDS-PAGE. A student determined a protein weighed 1200 kD via gel filtration, but the weight of the protein determined by SDS-PAGE indicated a weight of 300 kD. Why was there a discrepancy between the two weights for the protein? a. the student added four times as much (a higher concentration of proteins when he did gel filtration, hence a greater indicated weight for the protein b. the student forgot to add a reducing agent in his gel filtration, so the subunits of the protein are still linked with disulfide bonds, yielding a higher weight c. the protein used for gel filtration was contaminated by other proteins, leading to a higher weight count d. the protein the student was studying consisted of four identical subunits of 300 kD e. the student must have accidentally switched proteins when carrying out the two experiments; there is only one weight if he experimented on one protein structure

c. the protein used for gel filtration was contaminated by other proteins, leading to a higher weight count d. the protein the student was studying consisted of four identical subunits of 300 kD

In spectrophotometry, ___ nm wavelength absorption corresponds with ___ while the ___ nm wavelength absorption corresponds with protein. a. 260, Carbohydrate, 280. b. 280, Protein, 260. c. 260, DNA, 240. d. 260, DNA, 280. e. 240, Protein, 260.

d. 260, DNA, 280.

SDS-PAGE and agarose gel are two options in which proteins and DNA can be separated and analyzed. There are many differences and similarities between the two methods. Which of the following is NOT one of these similarities or differences? Select one: a. SDS is required when running protein samples with electrophoresis, since proteins need to be coated with a uniform, negative charge. b. Agarose is safe to handle as is, whereas acrylamide is safe to handle only after polymerization. c. In both gels, the samples run toward the positive electrode, since they are negatively charged. d. SDS-PAGE gels can only be used to run protein samples. Agarose gels can be used to run both protein and DNA samples. e. Agarose gel has a lower resolution than a SDS-PAGE gel.

d. SDS-PAGE gels can only be used to run protein samples. Agarose gels can be used to run both protein and DNA samples.

In the Polyacrylamide and Agarose Gels lab, when working with your protein or DNA sample you must wear: Select one: a. Gloves b. Lab coat c. Safety goggles d. Standard lab attire e. All of these

e. All of these

According to the lab safety sheet, which of the following is a potential hazard you will face in the Polyacrylamide and Agarose Gels lab? (Check all that apply) Select one: a. Water reactive chemicals b. None of these c. Carcinogenic chemicals d. Corrosive chemicals e. Chemicals causing respiratory tract, eye and skin irritation

e. Chemicals causing respiratory tract, eye and skin irritation

What chemical must be applied to proteins prior to running gel electrophoresis in order to denature the protein and give it an overall negative charge? Select one: a. Agarose b. EtBr c. Chelex d. Acrylamide e. SDS

e. SDS

Proteins denatured and coated with SDS all have an overall negative charge. The SDS coated proteins migrate at a rate relative to its ____, not its ____ and has ______ charge to mass ratio. a. charge only, shape and size, a different b. charge and shape only, size, the same c. size only, shape and charge, a different d. charge only, shape, a different e. size only, shape and charge, the same

e. size only, shape and charge, the same

what are two methods for estimating size of proteins?

gel filtration and SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

The distance a DNA molecule migrates during electrophoresis is:

inversely proportional to its size in base pairs

What are the functions of SDS in gel electrophoresis for estimating protein sizes?

it disrupts hydrogen bonding in proteins, linearizing the protein

Electrophoresis

method of separating serum proteins by electrical charge

SDS-PAGE acronym

sodium dodecyl sulfate polyacrylamide gel electrophoresis