Week 6: SDS

Agarose gels:

- Horizontal - Lower resolving power (50-20K bp) - Used for DNA quality/quantity measure after PCR - Stained with GelRed

Polyacrylamide gels

- Vertical, submerged in buffer - Higher resolving power (5-500 bp) - Used for protein subunit analysis and sequencing - Stained with Coomassie blue

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

A

Which of the following is NOT an important guideline to consider when designing primers? Select one: a. Presence of G or C at the 5' end. b. Length. c. Repeating bases in a row. d. Melting temperature. e. G-C content.

A

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

A

Which of the following is most affected by a mismatch in the 3' end of a primer with a DNA strand? Select one: a. Primer Efficiency b. AT% c. Primer Size d. Tm e. GC%

A

All of the following are true descriptions of agents involved in gel electrophoresis except

Agar is a stain which binds to proteins so that they may be visualized

What is BME used for in gel electrophoresis?

BME reduces disulfide bonds in proteins, separating different subunits

Which of the following is not considered to function as an accessory pigment in terms of absorption at a specific wavelength as it relates to their overall function?

Bacteriochlorophyll because they will receive the incoming photon, absorbs particular wavelengths of visible light, and converts the energy into another form

TLC separates pigments in all the ways except

By wavelength and the color that each pigment expresses.

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

When analyzing a DNA solution with spectrophotometry, which information can you get? Select one: a. the size of the DNA segment b. a qualitative measure of the DNA purity c. a quantitative measure of the concentration of DNA in the solution d. the sequence of the DNA e. the age of the DNA

C

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

Coomassie blue

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 agarose gel was run horizontally instead of vertically. b. The agarose gel is non-toxic, so we do not need SDS and beta mercaptoethanol. c. The PCR product is so small that SDS and beta mercaptoethanol are not needed. d. The PCR product is already linear and has a negative charge associated with it. e. The GelRed acted as a reducing agent and gave the PCR product a negative charge.

D

What is the commonly used unit to describe the size of a protein introduce din this lab?

Dalton/kilodalton

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 has only 1 subunit because both runs yield a single band. b. The protein contains no disulfide linkages. c. The consists of 2 subunits of equal size connected by a disulfide linkage. d. The protein contains only covalent bonds and no non-covalent interactions. e. The consists of 8 subunits of equal size connected by a disulfide linkage.

E

What information can gel filtration provide that is different from SDS-PAGE? Select one: a. The number of covalent bonds. b. The primary structure of a protein. c. The amount of noncovalent interaction in the protein. d. The weight of each subunit. e. The weight of a protein in its intact and functional form.

E

Which of the following is TRUE regarding analyzing protein sizes? Select one: a. smaller fragments are slower and do not travel as far as larger fragments b. protein fragments move down a gel from the positive end to the negative end. c. gel filtration is a technique that relies on breaking disulfide bonds to form peptide fragments d. The entire, complete protein moves down a gel from the negatively charged end to the positively charged end. e. polyacrylamide refers to the gel matrix in which the proteins migrate.

E

Which one of the following statements is false?

FITC denatures proteins and creates a uniformly negative charge in the polypeptide.

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

False

IN THIS LAB, you will use Coomassie Blue stain to visualize your protein on the gel. true or false?

False because we used AcquaStain in the lab, even though Coomassie Blue stain that is commonly used.

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

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.

Which of the following statements is true regarding visible spectrophotometry?

It is based on the fact that radiation in the wavelength range of 400-700nm can excite electrons in certain types of chemical bonds, elevating their energy levels away from the ground state.

________ is how energy is stored and released; ___________ is the storage of energy, ___________is the release of energy.

Metabolism, anabolism, catabolism.

If a protein that weighs 160 KD and has two subunits of different weights (60KD and 40KD) that are held together by disulfide linkages, is treated with SDS and a reducing agent, and run through gel electrophoresis, which of the following answer choices would resemble the gel picture?

One stronger band nearer to the top and one weaker band nearer to the bottom.

Which one of these statements is false?

PAGE gels can only be run vertically.

What kind of gel medium is used in SDS-PAGE?

Polyacrylamide

___________ is made of ___________, which is a neuron toxin and is safe only after polymerization. In contrast, the _________ in __________ is safe to use and extracted from seaweed.

Polyacrylamide gel, acrylamide, agarose, agarose gel.

You are conducting a gel electrophoresis to determine small differences in protein subunit molecular weights. Which of the following is a valid reason to choose a polyacrylamide gel over an agarose gel for your purposes?

Polyacrylamide has a higher resolution than agarose and will be able to distinguish smaller differences in sizes.

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

Quantitative

The combination of gel filtration and SDS-PAGE can reveal the ______ structure of a protein.

Quaternary

Which method will you use in this lab to separate proteins based on size? a) RASMOL b) SDS-PAGE electrophoresis c) gel filtration d) agarose gel electrophoresis e) none of these

SDS-PAGE electrophoresis

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

TGS (tris-glycine-SDS) buffer Tris glycine polyacrylamide gels

All of the following is true about Thin Layer Chromatography except:

The more soluble something is, the less it travels on the TLC.

An Rf value of 1, in the photosynthesis lab, would best be interpreted as

The pigment that travels best with the solvent.

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?

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

How were the proteins in the SDS-PAGE lab visualized?

The proteins were linked to a fluorescent tag and could be seen under a UV light.

Which of the following methods is NOT a way to separate proteins based predominantly on their size?

Thin layer chromatography.

In which situation would it be necessary to use an SDS-PAGE gel instead of an agarose gel?

To run a sample that contains small size differences within the sample.

Suppose after running a thin layer chromatography experiment with the same setup as in the photopigments lab, the pigments appear to remain clustered near the original spotting position and cannot be correctly read. What measures could be taken to increase the distance that the pigments travel on the TLC plate?

Use a less nonpolar mobile phase.

What is a protein ladder used for?

a protein ladder, DNA ladder, are markers that are used to identify the approximate size of a molecule run on a gel during electrophoresis

SDS, which stands for sodium dodecyl sulfate, is an anionic _______; it may break down _______ bonds.

detergent; dipole-dipole

In thin layer chromatography, a pigment with an Rf value equal to 0.98 would be:

found very near the solvent front.

Agarose gel electrophoresis as used in the lab you will be performing this week can do all of the following except: a) confirm the presence of purified DNA visually b) shower whether the DNA is the correct size 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

give a sequence for the amplified DNA

According to the equation that was created from the standard curve in SDS-PAGE lab, as the x-value ____, the y-value ____.

increases; decreases

The reducing agent, b-mercaptoethanol, in SDS-PAGE

reduces disulfide bonds which denatures the protein.

What is SDS (sodium dodecyl sulfate) used for in gel electrophoresis?

unfolds proteins and gives it an overall negative charge.