Lecture 5 Gel Electrophoresis

Question 1

Agarose Gel Electrophoresis

Answer 1

Analysis of nucleic acids and proteins
Separates molecules on basis of rate of movement through a gel under influence of an electrical field
Polymerized agarose is porous, allowing for movement of DNA. -meshlike so smaller fragments go through fast and will be in lower part of gel

Question 2

Principles of Nucleic Acid Seperation

Answer 2

Nucleic acids are neg charged because of sugar-phosphate backbone
Attracted to positive charged electrode

Question 3

Resolving Limits of Agarose Gels

Answer 3

0. 3% efficient range of separation 5-60 kb

0. 6% is 1-20kb

Question 4

Factors Affecting Nucleic Acid Migration

Answer 4

DNA or RNA molecular weight - smaller = faster
Voltage - higher the voltage, the faster the DNA moves
Buffer composition - TAE has the lowest buffering capacity but provides the best resolution for larger DNA

Question 5

Agarose Gel Applications

Answer 5

Estimation of the size of DNA molecules and restriction enzyme digestion pattern
Analysis of PCR products

Question 6

Agarose Advantages

Answer 6

Easily poured gel
Doesn’t denature sample, they can be recovered
Physically firmer than polycacrylamide

Question 7

Agarose Disadvantages

Answer 7

Gels can melt
Buffer can become exhausted
Different forms of genetic material may run in unpredictable forms
Lower bp resolution

Question 8

Polyacrylamide Gel

Answer 8

Very high resolution of DNA 10-3000 bp long Under the appropriate conditions, DNA molecules differing in size by only a single bp can be resolved

Question 9

SDS PAGE - Western Blotting

Answer 9

Western blot allows to determine the molecular weight of a protein and to measure relative amounts of the protein in different samples
Separate proteins according to their electrophoretic mobility or according to their size and no other physical feature. The enviro that allows separation by size is polyacrylamide

Question 10

SDS-PAGE Electrophoresis

Answer 10

SDS is neg charged detergent
Disrupts sec and tertiary structure by breaking H and unfolding protein
Masks charges on proteins so all have same charge
Prevents protein aggregation

Question 11

Western Blotting

Answer 11

Expression of protein of interest from a cell line of a tissue
Lyse cells
Centrifuge to remove debris
Analyze by SDS-PAGE

Question 12

3 Types of Western Blotting Visualization

Answer 12

1. Commassie brilliant blue - gives neg charge to protein. Not very sensitive, COMPATIBLE with mass spec
2. Silver stain - NOT compatible with mass spec, sensitive
3. Ponceau S - Limited sensitivity and is reversible method

Question 13

Western Blot - Transfer

Answer 13

Proteins usually seperated by SDS-PAGE gel
Then transferreto to sheet of special blotting paper called nitrocellulose or PVDF
Proteins retain same pattern of association they had on gel

Question 14

Immunoprecipitation

Answer 14

Same as western, just have agarose beads. Pull beads down, wash and remove protein. Then load gel, only specific protein is shown
Antibodies are used to isolate the proteins against which they are directed
Cell lysates are incubated with an antibody, which binds to its antigenic target protein

Question 15

Steps of Immunoprecipitaion

Answer 15

1 Cell nucleus containing the two interacting proteins of interest
2 Nuclear extraction
3 Add antibody directed against one of the proteins of interest
4 Add antibody binding beads
5 Immunoprecipitate the proteins of interest
6 Wash and collect the precipitated proteins

Question 16

Western Blot Detection

Answer 16

HRP (horseradish peroxidase) - catalyzes the oxidation of substrates by hydrogen peroxide, resulting in color or fluorescent product or release of light
AP (alkaline phosphatase) - catalyzes the hydrolysis of phosphate groups from a substrate molecule, resulting in a colored or fluorescent or release of light

Question 17

Western Blot Applications in Medicine

Answer 17

Confirm HIV, Hep B
Definitive test for mad cow disease
With ELISA diagnose Lymes

Question 18

ELISA

Answer 18

Antibodies can be used to quantify the amount of an antigen in a sample mixture
Rapid, convenient, detects less than a nanogram of a protein
If antigen is present, antibody-enzyme complex will bind to it

Question 19

Indirect ELISA

Answer 19

The production of color indicates the amount of an antibody to a specific antigen

Question 20

Sandwich ELISA

Answer 20

Production of color indicates quantity of anigen

Question 21

Western Blot

Answer 21

Confirm ELISA results
More specific
Identifies proteins by both antibody specificity and size

Question 22

ELISA

Answer 22

Quick results
Primary screening
Identifies proteins by antibody specificity only
Can give false positives, so has to be validated