Lab 6: Gel Electrophoresis
What is the optimum wavelength of UV light absorbed by protein?
280
What is the name of the method for separating charged molecules, such as proteins and nucleic acid, in an electrical field?
Electrophoresis
What is the purpose of the reducing agent, B-mercaptoethanol in SDS- PAGE?
It breaks disulfide linkages.
A student has obtained an unknown tertiary protein. Which of the following can the student do to determine both the molecular weight of the native protein and the total number of subunits in the protein?
Perform gel filtration and run a SDS-PAGE after mixing the protein with b-mercaptoethanol
When ejecting a protein sample into a well of the SDS-PAGE gel with a pipetter, why should you only push down on the pushbutton to the first stop and not the second stop?
Pushing down to the second stop will result in ejection of air from the pipet tip, which could blow the content intended for that particular well to the surrounding wells
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.
SDS is anionic and at high concentrations it binds to every side chain of proteins, disrupting ionic and hydrogen bonds, while reducing agents such as b-mercaptoethanol disrupt covalent bonds
You are in the lab trying to identify Protein X. The first gel you run for Protein X gives you one band at 30 kD and another band at 75 kD. You run another gel for Protein X using another technique. This time, you get one band at 210 kD. Circle below the possible techniques you used to obtain the above results.
SDS plus reducing agent in the first gel & SDS alone in the second gel
Which method will you use in this lab to separate proteins based on size?
SDS-PAGE electrophoresis
What is a difference between SDS-PAGE and agarose gel electrophoresis? a. Agarose gel electrophoresis separates on the basis of size while SDS-PAGE separates on the basis of charge. b. Agarose gel electrophoresis can only run DNA and SDS-PAGE can only run proteins. c. Agarose gel electrophoresis can only run proteins and SDS-PAGE can only run DNa. d. SDS-PAGE must be run vertically while agarose gel electrophoresis must be run horizontally. e. SDS-PAGE provides higher resolution results due to smaller pores.
SDS-PAGE provides higher resolution results due to smaller pores.
Which of the following is a difference between doing SDS-PAGE and using Agarose gel? a. Only SDS can be done vertically b. Only protein mass can be estimated by Agarose c. Only Agarose can be done vertically d. SDS-PAGE tends to be more accurate. e. Only protein mass can be estimated by SDS
SDS-PAGE tends to be more accurate
In Agarose gel electrophoresis, a. The gel is set up to run horizontally b. Agarose is toxic c. The gel is set up to run vertically d. The longest DNA fragments run the fastest towards the bottom e. We add SDS to coat DNA with a negative charge
The gel is set up to run horizontally
What is the MOST likely reason for a 1000 kD protein to have just one band on its SDS Page gel pattern? The protein is made up of 2 subunits, 750 and 250 kD, respectively.
The two subunits, connected by disulfide bonds, created one band.
A particular 4-subunit protein contains 3 identical subunits and 1 unique subunit which are all connected via non-covalent bonds. "SDS-PAGE analysis of this 4-subunit protein should yield how many bands?"
2
If a heterodimer with two different types of subunits of different sizes are treated with Beta-mercaptoethanol and run on an SDS-PAGE electrophoresis, How many band(s) would one expect?
2
After running an SDS Page gel you find 2 bands. The first band is 20kD in size and the second is 50kD. You know your protein mass is 140kD.How many subunits of each protein type do you have?
2 Subunits of 50kD and 2 Subunits of 20kD
What is the optimum wavelength of UV light absorbed by DNA?
260
Gel filtration determined the total size of a protein to be 230 kD. When a SDS polyacrylamide gel electrophoresis was run, there was one band at 70 kD and another at 40kD. Based on this, how many subunits might make up this protein?
5
In the Polyacrylamide and Agarose Gels lab, when working with your protein or DNA sample you must wear:
All of these (gloves, standard lab attired, lab coat, and googles)
SDS-PAGE functions to denature a protein by coating it in negative charges. Which of the following molecular interactions would not be disrupted by SDS-PAGE? a. Disulfide bridges b. Ionic interactions c. Van der Waal's Forces d. Hydrophobic interactions e. Hydrogen bonding
Disulfide bridges
SDS electrophoresis uses an ________ field to separate charged molecules & the SDS specifically disrupts __________ interactions?
Electric, noncovalent
After running electrophoresis in an agarose gel, it is treated with ethidium bromide for visualization. Which of the following best describes the action of Ethidium bromide on DNA?
Ethidium bromide inserts itself between the base pairs of DNA and can then be visualized under UV light.
Both SDS-PAGE gel and agarose gel were used in Lab 1 and 4 respectively. What is agarose gel treated with to visualize the bands on the gel under the UV light, and why does the treatment require safety attention?
Ethidium bromide; it is a suspected carcinogen
In this lab, the purpose of spectrophotometry is to measure protein activity.
False
SDS can break the covalent bonds that hold protein subunits together
False (SDS breaks noncovalent interactions)
In this lab, you will use Coomassie Blue to visualize your protein on the gel.
False (we use AcquaStain)
How does the method of gel filtration differ from that of SDS-Polyacrylamide gel electrophoresis(SDS-PAGE)?
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.
Why would a scientist choose to use polyacrylamide instead of agarose for gel electrophoresis?
He wants to separate DNA with very small size differences.
A student used the SDS-PAGE method to estimate the size of a protein. Through gel filtration the molecular weight of the protein was 400 daltons. However, when he did SDS-PAGE he saw 3 different lines at A (25 daltons), B (50 daltons) and at C (75 daltons). What is the total composition of the protein, including each subunits molecular weight? I. 2 of subunit A, 4 of subunit B, and 2 of subunit C II. 6 of subunit B, and 4 of subunit A III. 8 of subunit A, 3 of subunit B, and 2 of subunit C
I & III
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 gel 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
I and III
Which of the following statements is TRUE concerning the detergent SDS? a. For every two amino acids, an average of four SDS molecules associate with them. b. In a multisubunit polypeptide, it breaks apart disulfide linkages. c. It helps separate protein subunits by their charges. d. It coats proteins with a negative charge, with equal amount of charge per unit length. e. smaller protein subunits associate with more SDS molecules than do larger subunits.
It coats proteins with a negative charge, with equal amount of charge per unit length.
DNA is ___________ in order to run gel electrophoresis.
Linear and doesn't need to be treated with SDS
Which of the following does SDS disrupt between subunits of proteins? a. Noncovalent interactions. b. Hydrogen Bonds. c. Ionic Bonds. d. Covalent Bonds. e. Disulfide Bonds.
Noncovalent interactions.
After running a SDS-PAGE assay on an unknown protein with molecular size 180 kD, you find that the protein shows up as two bands on the polyacrylamide gel which correlate with a 30 kD subunit and a 50 kD subunit. What is a possible ratio of 30 kD subunits to 50 kD subunits that could constitute this mystery protein?
One 30 kD subunit: Three 50 kD subunits
What is the difference between SDS's function and reducing agent's function in SDS-PAGE?
SDS breaks the non covalent bonds and reducing agent breaks the disulfide bonds
Which one of the following statements about the SDS-PAGE technique used in the SDS-PAGE lab is incorrect? a. To make sure all subunits are dissociated, an agent that breaks disulfide linkages should be used. b. SDS causes proteins to unfold by disrupting beta sheets and alpha helices. c. SDS gives each polypeptide the same mass-to-charge ratio, which allows the polypeptides to be separated by mass. d. Polyacrylamide gel electrophoresis, like agarose gel electrophoresis, allows the smaller molecules to travel faster. e. SDS is a detergent that breaks covalent bonds.
SDS is a detergent that breaks covalent bonds.
We needed to determine the concentration of the DNA sample which we were analyzing. By what method were we able to measure this DNA concentration?
Spectrophotometry
In the agarose gel electrophoresis lab, why did we not add SDS and beta mercaptoethanol to the agarose gel before loading our PCR product?
The PCR product is already linear and has a negative charge associated with it.
Which of the following statements is not true regarding SDS-PAGE and agarose gel? a. Brightness, as a result of SDS-PAGE, might not be even; however, brightness, as a result of agarose gel, is an indication of DNA molecule size. b. Both SDS-PAGE and DNA agarose gel migrate toward the positive pole based on size. c. SDS-PAGE and agarose gel are used for both proteins and DNa. d. SDS-PAGE creates higher resolution than agarose gel. e. The fluorescent tag, FITC, is covalently bound to proteins to visualize them in agarose gel.
The fluorescent tag, FITC, is covalently bound to proteins to visualize them in agarose gel.
Which of the following is NOT TRUE regarding SDS-Page? a. The heaviest molecule travels the furthest down the gel b. SDS stands for "sodium dodecyl sulfate" c. molecular weight is estimated d. most common type of electrophoresis applied to proteins e. is also referred to as denaturing electrophoresis
The heaviest molecule travels the furthest down the gel
Which of the following statements is true? a. Discrepancies in the molecular weight estimates from SDS-PAGE and gel filtration indicates that the experiment was performed incorrectly as these two measures should be equal. b. Some proteins contain more than one subunit composed of identical polypeptide chains or different polypeptide chains, heterodimer or homodimer chains, respectively. c. SDS-PAGE provides an estimate of the molecular weight of the protein in its native state. d. The native state of a protein refers to the functional, intact form. e. The forces that stabilize subunits include the interaction of closely packed nonpolar and polar sidechains, hydrogen bonds, and disulfide bonds.
The native state of a protein refers to the functional, intact form.
How were the proteins in the SDS-PAGE lab visualized?
The polypeptides were covalently bound to a fluorescent tag that made them visible under UV light.
SDS-PAGE was performed on a sample treated with a reducing agent, and the resulting gel contains one band. Upon comparing this band to the ladder values, you determine that the band has a molecular weight of 200 kD. Which of the following is NOT possible? a. The protein is composed of two subunits, each with a molecular weight of 200 kD. b. The protein is composed of four subunits, each with a molecular weight of 50 kD. c. The protein is composed of four subunits, each with a molecular weight of 200 kD. d. The protein has a total molecular weight of 800 kD. e. The protein has a total molecular weight of 200 kD.
The protein is composed of four subunits, each with a molecular weight of 50 kD.
Which of the following is true about the SDS-PAGE and agarose gels? a. Using an agarose gel provides a higher resolution image than using SDS-PAGE. b. SDS-PAGE gels can only be used for DNA, whereas agarose gels can be used for either DNA or protein. c. Agarose gels can only be run horizontally, whereas SDS-PAGE gels can be run either horizontally or vertically. d. There are no toxic mutagens used in casting an agarose gel. e. Ethidium bromide is only used in running an SDS-PAGE gel.
There are no toxic mutagens used in casting an agarose gel.
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)?
There must be three 30kD subunits and three 40kD subunits.
Sally is running her SDS-PAGE forgets to add B-mercaptoethanol. The next day, she then runs her experiment again, this time using B-mercaptoethanol. How would the two gels differ?
They might look the same, depending on the presence of disulfide linkages within the protein
Why are having marker proteins of a known weight important in running an SDS-Page Gel along other proteins that have ambiguous weights?
They provide a comparison for the subunits whose weights are not known
Which of the following methods is NOT a way to separate proteins based predominantly on their size? a. SDS-PAGE. b. Electrophoresis. c. Gel filtration. d. Centrifugation. e. Thin layer chromatography.
Thin layer chromatography.
How was the molecular weight of the protein subunits determined in the SDS-PAGE lab?
Through constructing a standard curve.
What is the function of sodium dodecyl sulfate in SDS-Page?
To disrupt the noncovalent interaction between subunits of proteins, then adding an overall negative charge to the protein in proportion to its length
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
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
The average amount of SDS associated with a protein is estimated to be about _____ amino acid residue(s) per molecule of SDS
Two
After running an electrophoresis experiment, various methods are used to visualize the electrophoresed molecules. In the SDS-PAGE lab, what method of visualization was used to allow us to see the protein bands?
UV illumination alone since the proteins were already pre-labeled with FITC, a covalently attached florescent tag.
What other property was accounted for along with shape in running proteins in the SDS-PAGE lab?
charge
What is the commonly used unit to describe the size of a protein introduced in this lab?
dalton/kilodalton
Dithiothreitol has the same function as b-mercaptoethanol when used in SDS-PAGE, which serves to ________
denature proteins by disrupting a type of covalent linkage between protein subunits.
In the SDS-PAGE lab, BME (b-mercaptoethanol) is a reagent that:
reduces disulfide bonds.
In SDS-PAGE, the movement of polypeptides through the gel will be affected by polypeptide
size
When proteins are separated with SDS-PAGE, the ______ subunits migrate the _______ from the ______ electrode to the ______ electrode on each side of the polyacrylamide gel.
smallest, fastest, negative, positive
In the Agarose Gel Electrophoresis and Molecular Clocks lab, we analyze a Polymerase Chain Reaction (PCR) product by using _____________.
spectrophotometry to provide a quantitative measure of the concentration of DNA
In the Agarose Gel Electrophoresis lab, we analyze a Polymerase Chain Reaction (PCR) product by using _____________.
spectrophotometry to provide a quantitative measure of the concentration of DNA
Some of the chemicals/materials you will use in the Polyacrylamide and Agarose Gels lab include (check all that apply):
-TGS (tris-glycine-SDS) buffer -Tris glycine polyacrylamide gel
How many bands can one expect to see on an SDS-Page gel, if a protein is a homotetramer?
1
In the SDS-PAGE lab, a student decides to run a human hemoglobin protein which has 4 identical subunits. If he runs one sample with just SDS, he will see _____ band(s), and if he runs it with a reducing agent he will see _____ band(s).
1 band; 1 band
If an SDS-PAGE gel photograph shows protein subunit bands at 40kD and 60kD, and you know that the total weight of your unknown protein is 240kD, you know that you would have ____ of the 40kD subunit and ____ of the 60kD subunit. From this, you also know that your unknown protein is a __________.
3; 2; heterodimer
In SDS-PAGE electrophoresis, SDS has all of the following functions EXCEPT: a. Disrupts noncovalent interactions between subunits b. Associates with unfolded protein backbone c. Causes polypeptide chains to unfold d. Breaks disulfide linkages e. Conferring overall negative charge to protein
Breaks disulfide linkages
What does an agarose gel electrophoresis NOT confirm.
Concentration of DNA (it confirms DNA size based on standard curve, Relative intensity of DNA, Presence of DNA, DNA composed of primer-dimer)
Which of these substances is usually used to visualize proteins on a polyacrylamide gel
Coomassie blue
Which of the following statements is true? a. DNA must be treated with SDS in order to run it on a gel. b. The electromotive force drives the negatively charged DNA toward the cathode during DNA gel electrophoresis. c. Agarose gels have a small range of separation and high resolving power. d. Polymerizing acrylamide makes it a neurotoxin. e. DNA gel electrophoresis is primarily employed after PCR is carried out.
DNA gel electrophoresis is primarily employed after PCR is carried out.
SDS denatures protein by all of the following means EXCEPT: a. Interfering with ionic interactions. b. Interfering with hydrophobic interactions. c. Disrupting disulfide bonds. d. Interfering with Van der Waals interactions e. Disrupting hydrogen bonds.
Disrupting disulfide bonds.
What would be a reason you would use an agarose gel as opposed to a polyacrylamide gel when running electrophoresis?
It is easy to make since it is nontoxic prior to polymerization and does not need to be made in airtight conditions
For SDS page, the ladder had multiple subunits because
It is used to identify the size of different subunits.
In this lab, an SDS-PAGE technique was performed to analyze protein subunits. What was the main function of polyacrylamide in this lab?
It was the gel matrix in which the proteins were able to migrate based on their size.
Which one of these statements is false? a. FITC can be used for visualization on a PAGE gel. b. PAGE gels can only be ran vertically. c. DNA travels towards the positive electrode in gel electrophresis. d. Ethidium bromide is highly toxic and carcinogenic. e. The smaller pores on a PAGE gel allow for greater resolution than on an agarose gel.
PAGE gels can only be ran vertically.
What kind of gel medium is used in SDS-PAGE?
Polyacrylamide
If you ran a DNA agarose gel, but you did not see any DNA products under UV. What is possibly a reason to this?
You forgot to put ethidium bromide in the gel, hence no fluorescence is seen under UV.
A protein is run through a SDS-PAGE and two bands are observed however, you suspect that disulfide bonds could still be holding individual subunits together, what would be the best course of action?
add an reducing agent to break the bonds
A protein is run through a SDS-PAGE and two bands are observed however, you suspect that disulfide bonds could still be holding individual subunits together, what would be the best course of action?
add an reducing agent to break the bonds
The detergent SDS was used for the analysis of proteins to:
assign a uniform negative charge throughout the polypeptide (to give them all an equal charge/mass ratio).
Why did we use an agarose gel instead of a polyacrylamide gel to determine if the PCR of our mitochondrial DNA had worked?
because high resolution was not needed since we were just looking for a band around 500bp.
During SDS-PAGE, the reducing agent breaks _______ bonds while the SDS creates an overall ____ charge on the protein. Thus, the proteins migrate toward the ___electrode on the gel.
disulfide, negative, positive
During SDS-PAGE, the reducing agent breaks _______ bonds while the SDS creates an overall_______ charge on the protein. Thus, the proteins migrate toward the _______ electrode on the gel.
disulfide, negative, positive
Which of the following techniques allows the determination of a protein's mass in its native form? a. ultracentrifugation b. RASMOL c. Western Blot d. gel filtration e. SDS-page
gel filtration
Agarose gel electrophoresis as used in the lab you will be performing this week can do all of the following except:
give a sequence for the amplified DNA
SDS-PAGE resolves polypeptides based on differences in
mass.
After the treatment of proteins described in the lab manual, all proteins used in this lab should carry an overall __________ charge.
negative
In lab, polypeptides were separated on a _____________ while DNA fragments were separated on a ____________________.
polyacrylamide gel; agarose gel
In lab, polypeptides were separated on a/an _____________ while DNA fragments were separated on a/an ____________________.
polyacrylamide gel; agarose gel
While running PCR products on an agarose gel, samples that are closer to the ___________ electrode are _________ in size.
positive, smaller
Which type of trendline is used to construct a standard curve from the distances traveled by the standard bands of a known marker in an SDS-PAGE experiment?
power
Spectrophotometry provides a ______ measure of the concentration of DNA
quantitative
The combination of gel filtration and SDS-PAGE can reveal the ______ structure of a protein.
quaternary
Which is NOT a function of SDS (sodium dodecyl sulfate) in SDS-PAGE? a. to eliminate the effect of shape on the determination of polypeptide size b. to disrupt weak intermolecular interactions. c. to produce a uniform charge: mass ratio among polypeptides d. to denature protein e. to allow visualization of the proteins under UV light
to allow visualization of the proteins under UV light
When running an SDS-PAGE gel, what is the purpose of the marker/ladder, SDS, and the reducing agent? a. to provide a standard of measure, to coat the protein with negative charges, to denature the protein b. to provide a standard of measure, to denature the protein, to coat the protein with negative charges c. to coat the protein with negative charges, to denature the protein, and to provide a standard of measure d. to coat the protein with positive charges, to denature the protein, and to provide a standard of measure e. to provide a standard of measure, to coat the protein with positive charges, to denature the protein
to provide a standard of measure, to coat the protein with negative charges, to denature the protein
Protein X contains 4 different sizes of subunits. Subunits A and B are connected by hydrogen bonds, while subunit C is connected to subunit A by disulfide bonds, and subunits D is also connected to B by disulfide bonds. Protein X was run through SDS-PAGE without b-mercaptoethanol showing _______ bands because SDS disrupts __________bonds, compared to ____ bands when X was treated with b-mercaptoethanol, as b mercaptoethanol disrupts __________bonds.
2, non-covalent, 4, disulfide
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 and II
A student has obtained an unknown tertiary protein. Which of the following can the student do to determine both the molecular weight of the native protein and the total number of subunits in the protein? a. Perform ultracentrufigation to determine the total mass of the native protein and use gel filtration to determine the number of subunits within in the protein b. Run a SDS-PAGE after mixing the protein with coomassie blue and perform a Western Blot c. Run a PAGE after denaturing the protein in SDS and perform ultracentrifugation through a glucose gradient using flourescein isothiocyanate (FITC) d. Perform gel filtration and run a SDS-PAGE after mixing the protein with b-mercaptoethanol e. Run a SDS-PAGE with coomasie blue and b-mercaptoethanol followed by X-ray crystallography
Perform gel filtration and run a SDS-PAGE after mixing the protein with b-mercaptoethanol
Which one of the following is not true about polyacrylamide gel and/or Agarose DNA gel? a. In both Agarose and polyacrylamide gel, the brightness under the UV light depends on abundance of the molecule b. They have different resolution c. Agarose and polyacrylamide gel can be both vertical and horizontal d. Polyacrylamide is used only for protein but Agarose gel can be used for both DNA and proteins e. SDS in polyacrylamide gel denatures the protein and add negative charge to them.
Polyacrylamide is used only for protein but Agarose gel can be used for both DNA and proteins
Select the false statement: a. the reducing agent breaks down disulfide bonds. b. bands closer to the loading wells are indicative of 'heavier' protein fragments. c. the protein fragments travel to the cathode end of the gel matrix. d. SDS is responsible for distributing an overall positive charge to the protein fragments at an equal charge to mass ratio enabling them to traverse through the negatively-charged gel. e. the acronym PAGE stands for 'polyacrylamide gel electrophoresis'.
SDS is responsible for distributing an overall positive charge to the protein fragments at an equal charge to mass ratio enabling them to traverse through the negatively-charged gel.
Which of the following statements is INCORRECT? a. SDS-PAGE provides data on the mass of a protein in its native (intact) state. b. SDS-PAGE provides information to estimate the correct size of the protein in number of basepairs. c. SDS-PAGE eliminates effects of protein shape on the determination of protein mass. d. SDS-PAGE provides information about the mass of a protein that is complimentary to that determined by gel filtration. e. SDS-PAGE provides information about the quaternary structure of a protein.
SDS-PAGE provides data on the mass of a protein in its native (intact) state.
How were the proteins in the SDS-PAGE lab visualized? a. The gel was stained with coomassie blue and the proteins could be seen under a UV light. b. The proteins were tagged with ONPG and could be detected with the spectrophotometer. c. The proteins were stained with ethidium bromide and could be seen under a UV light. d. The proteins were linked to a fluorescent tag and could be seen under a UV light. e. The gel was stained with ethidium bromide and the proteins could be seen under a UV light.
The proteins were linked to a fluorescent tag and could be seen under a UV light.
