Polyacrylamide and Agarose Gel Electrophoresis Lab F

In this lab, the purpose of spectrophotometry is to measure protein activity. True or False

False

In this lab, you will use Coomassie Blue to visualize your protein on the gel. True or False

False

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

False

According to the lab safety sheet, in the Polyacrylamide and Agarose Gels lab, used microcentrifuge tubes and pipette tips should be disposed of in the regular trash. True or False

True

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

True

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

Spectrophotometry provides a ______ measure of the concentration of DNA. Select one: a. quantitative b. qualitative

a

What is the optimum wavelength of UV light absorbed by DNA? a. 260 b. 280 c. 320 d. 680 e. 570

a

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

What are the functions of SDS in gel electrophoresis for estimating protein sizes? choose all that apply a. Disrupts H bonding in proteins, linearizing proteins b. Provides an overall negative charge on proteins, making the migration distance on gel a function of only protein size c. Intercalates between the amino acids of the protein allowing it to be visualized on the gel

a, b

A scientist runs an SDS-PAGE experiment two separate times on a protein with a molecular weight of 200 kD. For the first experiment he treats the protein with SDS only and for the second experiment he treats the protein with both SDS and a reducing agent. If the first gel yields one band showing a molecular weight of 200 kD and the second run yields one band showing a molecular weight of 25 kD, the researcher should conclude that Select one: a. The protein contains no disulfide linkages. b. The consists of 8 subunits of equal size connected by a disulfide linkage. c. The protein has only 1 subunit because both runs yield a single band. d. The consists of 2 subunits of equal size connected by a disulfide linkage. e. The protein contains only covalent bonds and no non-covalent interactions.

b

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

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

b

All of the following is true about agarose gels EXCEPT: Select one: a. Conventionally, agarose gel electrophoresis is set up horizontally. b. Agarose gel electrophoresis has a higher resolution than SDS-PAGE. c. The pores in agarose gels are bigger than those in polyacrylamide gels. d. Agarose is safe to handle, whereas acrylamide is a neurotoxin. e. Agarose gel electrophoresis can be used for the separation of DNA fragments.

b

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

In the Gel Electrophoresis lab, why did we not add SDS and beta mercaptoethanol to the agarose gel before loading our PCR product? Select one: a. The GelRed acted as a reducing agent and gave the PCR product a negative charge. b. The PCR product is already linear and has a negative charge associated with it. c. The agarose gel was run horizontally instead of vertically. d. The agarose gel is non-toxic, so we do not need SDS and beta mercaptoethanol. e. The PCR product is so small that SDS and beta mercaptoethanol are not needed.

b

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

b

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

What is the name of the method for separating charged molecules, such as proteins and nucleic acids, in an electrical field? a. chemophoresis b. electrophoresis c. electroportation d. denaturation e. electroseparation

b

Which of these substances is usually used to visualize proteins on a polyacrylamide gel? a. Ethidium bromide b. Coomassie blue c. Aniline blue d. Phenol red e. Alcian blue

b

An antibody, with molecular weight 90 kD in its native state, is treated with SDS and a reducing agent before being loaded onto an SDS-PAGE gel. The result shows 2 bands, one is 20 kD and the other band is at 50 kD. How many subunits are in this antibody? Select one: a. 1 b. 2 c. 3 d. 4 e. 5

c

Which of the following is a potential hazard you will face in the Polyacrylamide and Agarose Gels lab? a. corrosive chemicals b. carcinogenic chemicals c. chemicals causing respiratory tract, eye, and skin irritation d. water reactive chemicals e. none of the above

c

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

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. Chloroform b. Ethidium bromide c. Tris glycine polyacrylamide gels d. TGS (tris-glycine-SDS) buffer e. none of these

c, d

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

d

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

d

What is the optimum wavelength of UV light absorbed by protein? a. 570 b. 680 c. 260 d. 280 e. 320

d

What is the purpose of the reducing agent, B-mercaptoethanol in SDS- PAGE? Select one: a. It allows the proteins to travel more quickly through the SDS-Polyacrylamide gel. b. It separates the individual subunits of the protein, on the basis of differences in their molecular weights, using polyacrylamide gel electrophoresis (PAGE). c. It denatures proteins, disrupting noncovalent linkages between subunits (polypeptides). d. It breaks disulfide linkages. e. It associates with the unfolded backbone of the protein, conferring an overall negative charge to the protein in proportion to its length.

d