Lab F - Gel Electrophoresis
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's the optimum wavelength of UV light absorbed by DNA?
260 nm
nucleic acid absorbs light mostly at
260 nm
protein absorbs light mostly at
280 nm
Which of the following best describes the function of polyacrylamide gel used in SDS-PAGE? Select one: a. It separates proteins based on size due to the web-like matrix formed by its molecules. b. It binds to the protein bands and allows them to be seen under UV light c. It acts as the reducing agent which breaks down disulfide bonds within the protein. d. It contains enzymes that break down complex proteins into its major subunits. 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.
Which pores are bigger? Those in agarose gels or polyacrylamide gel?
Agarose gel
All of the following are true descriptions of agents involved in gel electrophoresis except: Select one: a. SDS is an anionic detergent that disrupts non-covalent interactions and confers an overall negative charge to the protein in proportion to its length. b. B-mercaptoethanol is a reducing agent that breaks disulfide linkages between and within polypeptide chains c. Agar is a stain which binds to proteins so that they may be visualized. d. GelRed is a less toxic alternative to the traditional ethidium bromide stain. e. Polyacrylamide is the gel matrix in which the denatured proteins migrate that is mounted between two buffer chambers containing separate electrodes creating an electrical connection through the gel.
C. Agar is a stain which binds to proteins so that they may be visualized.
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 __________. Select one: a. 4; 1; monomer b. 3; 2; tetramer c. 3; 2; homodimer d. 3; 2; pentamer Correct e. 2; 3; dimer
D. 3; 2; pentamer
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. Agarose is safe to handle as is, whereas acrylamide is safe to handle only after polymerization. b. SDS is required when running protein samples with electrophoresis, since proteins need to be coated with a uniform, negative charge. c. 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. e. 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.
What is the name of the method for separating charge molecules, such as proteins and nucleic acid, in an electrical field?
Electrophoresis
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 covalently bonds to DNA and can then be visualized under UV light. eb intercalates between dna base pairs and fluoresces under UV light, making it possible to easily see dna bands
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
in this lab, you will use coomassie blue to visualize your protein on gel.
False
The agarose gel used in this lab are casted with ____, a less toxic version of ____
Gel Red, a less toxic versoin os ethidium bromide
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
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? a. No bands b. One stronger band nearer to the top and one weaker band nearer to the bottom. c. One weaker band nearer to the top with one stronger band nearer to the bottom. d. One stronger band nearer to the top with two weaker bands nearer to the bottom. e. One band only
One stronger band nearer to the top and one weaker band nearer to the bottom.
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 to separate proteins based on size?
SDS-PAGE electrophoresis
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
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. Tris glycine polyacrylamide gels b. TGS (tris-glycine-SDS) buffer c. Ethidium bromide d. none of these e. Chloroform
Tris glycine polyacrylamide gels, TGS (tris-glycine-SDS) buffer
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
what is the difference between agarose gel and the polyacrylamide gel?
agarose was already stained with gelred so no need to stain it like we do with polyacrylamide with nublu
All of the following are differences between agarose gel electrophoresis and SDS-PAGE except a. In the polyacrylamide gel lab, FITC was used to visualize the samples, whereas in the agarose gel lab, ethidium bromide was used to visualize the gels. b. Agarose gels can only be used to detect DNA, whereas SDS-PAGE gels can be used to detect both protein and DNA. c. In the polyacrylamide gel lab, the samples needed to be treated with SDS before they could be analyzed, whereas in the agarose gel lab, no additional preparation needed to be done to the samples. d. In the polyacrylamide gel lab, the gel was run vertically, whereas in the agarose gel lab, the gel was run horizontally. e. Polyacrylamide gels are unsafe to use in their unpolymerized form, whereas agarose gels are safe enough to be eaten.
b. Agarose gels can only be used to detect DNA, whereas SDS-PAGE gels can be used to detect both protein and DNA.
SDS-PAGE and agarose gel electrophoresis are both laboratory techniques used in separating compounds by molecular weight. Which of the following is FALSE? a. Both techniques require a buffer solution. b. Both techniques can be used to separate proteins by size at reasonable resolutions. c. Compounds separated must have uniform linear shape. d. Both techniques can be used to separate DNA by size at reasonable resolutions. e. Compounds separated must carry an evenly distributed negative charge.
b. Both techniques can be used to separate proteins by size at reasonable resolutions.
What is the commonly used unit to describe the size of a protein introduced in this lab? Select one: a. gram/kilogram b. dalton/kilodalton c. none of these d. centimeters e. base/kilobase
b. dalton/kilodalton
why do smaller molecules move faster in agarose gels?
because the gel is a polymeric network effectively filled with pores, the smaller molecules move faster
In SDS-PAGE, why do big proteins pass through first?
because the small proteins are going through the spheres still
one the gel is imaged, how do we determine the sizes of unkown protein subunits?
by comparing the distances these polypeptides migrated to the standard curve constructed from the distances migrated by standard proteins that have known weights
which substance is usually used to visualize protein on polyacrylamide gel
coomassie blue b/c it stains gel and protein bands and requires time consuming de-staining step. for this lab we use nublu express
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
SDS-PAGE is a TYPE of electrophoresis that is known as the
denaturing electrophoresis
what are functions of sds in gel electrophoresis for estimating protein sizes?
disrupts h bonding in proteins, linearizing protein. provides overall negative charge on proteins making migration distance on gel function of only protein size
2 methods for estimating protein size
gel filtration and sds-polyacrylamide gel electrophoresis (sds-page), yield diff, but complementary data
when the absoroption ratio of 260nm/280nm is high, this indicates
good nucleic acid purity
Gel electrophoresis can for two things. what are they?
it can mesaure protein size and determine the quarternary structure --- can determine how many subunits a protein has, the size of each subunit, and how many of each type there are
What is the purpose of SDS?
it disrupts the noncovalent linkages
what is the purpose of gel filtration
it provides an estimate of the molecular weight of the protein in its native or intact state
what does polyacrylamide in PAGE refer to
it refers to the gel matrix in which the proteins migrate - its mounted b/w two buffer chambers containing separate electrodes so that the only electrical connection between the two chambers is the gel
After treatment, all proteins should carry a ____ charge
negative
rate of molecule's migration thru electrical field depends on net charge, size, shape of molecules and strength of electrical field
net charge, size, shape of molecules and strength of electrical field
How are new amino acids added to other amino acids?
new amino acids are added to the carboxyl end of previous amino acid
Electrophoresis confirms.....
presence of purified DNA visually and gives qualitative overview of DNA purity
Spectrophotometry provides a ____ measure of DNA concentration
quantiitative
The reducing agent, b-mercaptoethanol, in SDS-PAGE
reduces disulfide bonds which denatures the protein
when is the electrophoresis complete?
since its based on size - electrophoresis is complete once the smallest and fastest protein reaches in the end of the gel
What are proteins separated by in column chromatography?
size, hydropohobicity, charge, or binding to specific group
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
Why does DNA move through the agarose gel
the negatively charged DNA moves towards the positive electrode
what is the purpose of the SDS-polyacrylamide gel electrophoresis?
the purpose is to find the number of subunits in the protein - the detergent SDS denatures the proteins, disrupting the noncovalent linkages between subunits - used with a reduing agent that breaks disulfide linkages, the indiivudal subunits of the pproteins can be separated on the basis of differences in their molecular weights using PAGE (polyacrylamide gel electrophoresis
The combo of gel filtration and SDS-PAGE can reveal ____ structure of a protein
the quaternary structure
an electric field is applied so that...
the sample migrate from the negative side to the positive side
What is the purpose of beta-mercaptoethanol?
this is the reducing agent that breaks the disulfide linkages
What is x-ray crystallography used for?
to find protein structure; uses protein crystal and x-rays to produce refraction patterns; e- density is calculated and then a wire model of a protein can be modeled into the density
agarose gels used int his lab are cat w/ gelred, a less toxic alternative to ethidium bromide.
true
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?
two
how do we visualize the SDS-PAGE gel after everything is done
we stain it with NuBlue Express to see the blue bands which can be visualized by camera
