An Introduction to SDS Page

Question 1

What is the name of this lab?

Answer 1

An Introduction to SDS Page

Question 2

What is electrophoresis?

Answer 2

To carry with electricity

Question 3

General Principles of Protein Electrophoresis and __________ is the migration of charged molecules in an electric field toward the electrode with the _____ charge.

Answer 3

SDS-PAGE Electrophoresis; the opposite

Question 4

What is the acronym SDS-PAGE stand for?

Answer 4

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Question 5

SDS-PAGE is a form of electrophoresis that treats samples with ____ to ____ proteins.

Answer 5

SDS; denature

Question 6

What value is there on the SDS-PAGE technique?

Answer 6

It helps find the:

• # proteins in a sample;
• molecular weights of proteins;
• differences in proteins from different sources;
• purity of protein of interest;
• amount of protein present

Question 7

Proteins are usually separated using agarose gels. True or False?

Answer 7

False. Proteins are separated using polyacrylamide gels. (Agarose gels are used to separate DNA.)

Question 8

Proteins are usually separated using polyacrylamide gels. True or False?

Answer 8

True

Question 9

Why are proteins separated by polyacrylamide gels?

Answer 9

Most proteins are much smaller than DNA fragments AND polyacrylamide gels have pore sizes similar to the sizes of proteins.

Question 10

The gel matrix formed by polyacrylamide is much ______ than agarose and able to resolve much _____ molecules.

Answer 10

tighter; smaller

Question 11

Polyacrylamide gels are often used to separate very _____ molecules (_____ bp) for DNA sequencing or PCR analysis, while agarose is sometimes used to separate very ____ molecules.

Answer 11

small; <500 bp; large

Question 12

PAGE uses how many phases of polyacrylamide?

Answer 12

2

Question 13

The two phases of polyacrylamide used are the upper stacking gel typically of __% acrylamide and a lower resolving gel of a ____ percentage of acrylamide.

Answer 13

4; higher

Question 14

This is called a _______ system and results in all of the proteins in a sample ______, or ______, at the same time.

Answer 14

discontinuous; separating; resolving

Question 15

Since sample volumes can vary from lane to lane, forming vertically narrow or broad bands in the wells, all of the proteins in a sample enter the gel simultaneously. True or False.

Answer 15

False, The proteins do NOT enter the gel simultaneously.

Question 16

The ___ percentage of the stacking gel allows the proteins to migrate _____ and be _____ at the edge of the denser resolving gel regardless of their sizes.

Answer 16

low; rapidly; compressed

Question 17

The samples of _________ are thus concentrated ____ thin bands in each lane before they move into the denser resolving gel and begin to be separated according to their ________.

Answer 17

mixed proteins; uniformly; molecular weights

Question 18

To establish the ion fronts, the SDS-PAGE running buffer is made with _____ and ____ (ph ___)….

Answer 18

Tris; glycine; 8.3

Question 19

The Ready Gel polyacrylamide gel is made with _____ (pH ____).

Answer 19

Tris-HCl; 8.8.

Question 20

Since ____ ions migrate more rapidly than ____ ions in an electric field, and proteins have intermediate mobility; the proteins become _____ in a narrow band between ion fronts when electrophoresis is begun.

Answer 20

chloride; glycine; trapped

Question 21

Polyacrylamide does not require a chemical reaction to cause polymerization of two acrylamide monomers. True or False?

Answer 21

False. They do require a chemical reaction.

Question 22

Polyacrylamide requires a chemical reaction to cause polymerization of two acrylamide monomers. True or False?

Answer 22

True

Question 23

What must be added to a solution containing the desired concentrations of acylamide and bis-acrylamide monomers in a Tris buffer to cast a polyacrylamide gel?

Answer 23

• a reaction inhibitor;
• ammonium persulfate (APS);
• catalyst
• tetramethylethylenediamine (TEMED)

Question 24

To cast a gel with a resolving and stacking gel of different polyacrylamide concentrations, a ____________ gel is poured first and the ____ concentration stacking ___ is poured on top of it.

Answer 24

high-concentration resolving; low; gel

Question 25

A _________ is inserted into the unpolymerized stacking gel solution.

Answer 25

sample comb

Question 26

The comb is removed after polymerization is complete to create ____ for _______.

Answer 26

wells; sample loading

Question 27

DNA is quantified in terms of _____.

Answer 27

length or # of base pairs

Question 28

Proteins are quantified in terms of _______ relative to a hydrogen atom, in ______.

Answer 28

molecular weight; Daltons

Question 29

DNA is composed of only ____ nucleotides, which are in roughly equal proportions and are roughly the same ______.

Answer 29

4; molecular weight

Question 30

Proteins are composed of ____ amino acids with molecular weights from ___ to ___ Daltons (the average is 110) and whose peptide chains vary considerably in percentage of amino acids.

Answer 30

20; 89; 204

Question 31

One Dalton equals the mass of a _____ atom, which is ________ grams.

Answer 31

hydrogen; 1.66x10^-24

Question 32

Most proteins have masses on the order of thousands of Daltons, so the term _____ is used for protein molecular masses.

Answer 32

kilodalton

Question 33

Proteins range in size from several kilodaltons to thousands of kilodaltons, but most fall between the range of ___ kD and ___ kD,

Answer 33

10; 220

Question 34

A molecule’s ____ and its _____ affect its mobility through a gel during ____.

Answer 34

electrical charge; mass; electrophoresis

Question 35

The ratio of charge to mass is called _____.

Answer 35

charge density

Question 36

The inherent charges of proteins must be removed as a factor affecting _______ in order for ________ ________ to be effective as a method of protein molecular weight determination.

Answer 36

migration; polyacrylamide electrophoresis

Question 37

What 2 things does SDS do?

Answer 37

• Binds to and coats the proteins;

* Keeps the proteins denatured as relatively linear chains

Question 38

In this form, protein migrate in a polyacrylamide gel as if they have equivalent negative charge densities, and ____ becomes the main variable affecting the migration rate of each protein.

Answer 38

mass

Question 39

Which protein structures of the protein complexes within a protein extract are disrupted prior to electrophoresis?

Answer 39

secondary, tertiary and quarternary

Question 40

The process of structural disruption is called ____.

Answer 40

denaturation

Question 41

Which structure is denatured linear chain of amino acids?

Answer 41

Primary

Question 42

Which structure depicts several polypeptide chains associated together to form a functional protein?

Answer 42

Quarternary

Question 43

Which structure depicts domains of repeating structures, such as Beta-pleated sheets and Alpha helices.

Answer 43

Secondary

Question 44

Which structure represents 3-dimensional shape of a folded polypeptide, maintained by disulfide bonds, electrostatic interactions and hydrophobic effects?

Answer 44

Tertiary

Question 45

Which reducing agents can be used to add to the samples to ensure complete breakage of disulfide bonds?

Answer 45

B-mercaptoethanol (BME) or dithiothreitol (DTT)

Question 46

What 3 factors completely disrupt the secondary, tertiary and quarternary structures?

Answer 46

heat
ionic detergent
reducing agent

Question 47

The molecules snake though the gel at rates proportional to their….

Answer 47

molecular masses

Question 48

Both BME and DTT are potentially _____ and produce _____ smell.

Answer 48

hazardous; unpleasant

Question 49

The protein banding patterns of ____ muscle tissues are not greatly affected by the exclusion of reducing agents, but the addition of a reducing agent to the _______ sample buffer does reduce background bands.

Answer 49

fish; Laemmli

Question 50

___ may be used at the discretion of the instructor for the protein profiler module.

Answer 50

DTT

Question 51

A quarternary protein complex denatured with _____, ______ and ____ can be separated into individual proteins and resolved by size using _____.

Answer 51

reducing agent; heat; SDS; SDS-PAGE

Question 52

The combination of ____ and the detergent ____ denatures proteins for _____ analysis.

Answer 52

heat; SDS; SDS-PAGE

Question 53

When identifying proteins in Polyacrylamide Gel, the different proteins appear as a distinct ____-stained bands on the finished stained gel.

Answer 53

blue

Question 54

From the positions and intensities of these bands; we can determine the _____ and relative ____ of the proteins, but we can only make educated guesses about the ____ of each protein.

Answer 54

size; abundance; identity

Question 55

The _____ and ______ standard helps to identify these proteins.

Answer 55

actin; myosin

Question 56

The _____________ prestained protein standards are used to determine the molecular masses of the unknown proteins and to help monitor the progress of the run.

Answer 56

Precision Plus Protein Kaleidoscope

Question 57

Definitive identification of protein requires mass ______, ______, or _______.

Answer 57

spectrometry; sequencing; immunodetection

Question 58

Immunodetection methods, such as ______, use ____ that specifically recognize the proteins of interest.

Answer 58

western blotting; antibodies

Question 59

What is the name of the experimental portion of the lab?

Answer 59

Electrophoresis: Gel loading and Running