Tools Of Biochemistry

Question 1

Gel Electrophoresis

Answer 1

DNA, Protein
Size and shape
SDS page denatures and reduces

Question 2

Western Blot

Answer 2

Shows antibody (synthetic) for specific protein (human)

1. Electrophoresis
2. Blot to paper
3. Antibody wash
4. Tag and fluoresce

Question 3

Affinity Chromatography

Answer 3

Looks for protein through affinity to fixed antibodies

1. Lyse cells, isolate proteins
2. Run through fixed column with antibodies
3. Elute fixed protein and fluoresce

Question 4

Epitope tagging

Answer 4

Looking for epitope’s antibodies, to discover protein

1. Attach epitope to protein
2. Wash with antibodies specific for epitope
3. Fluoresce

Question 5

ELISA Direct

Answer 5

Looking for primary or secondary antibodies

1. Electrophoresis
2. Antibody wash
3. Flouresce the antibody tag

Question 6

ELISA Indirect

Answer 6

Looking for secondary antibody

1. Electrophoresis
2. Antibody wash
3. Secondary antibody attachment
4. Fluoresce

Question 7

ELISA Use

Answer 7

Good for quantification of protein amount

Question 8

Immunohistochemistry

Answer 8

Done in situ
Used to see where proteins are present in tissue 
1. Wash tissue with antibody
2. Antibody binds to protein (antigen)
3. Secondary antibody attachment
4. Fluoresce

Question 9

Protein Microarrays

Answer 9

Analytical- protein levels
Functional- rate of protein interaction
1. Protein immobilized on chip
2. Washed with solution containing antibodies/substrates/labels
3. Fluoresce, yellow is the mixed color

Question 10

Restriction Enzymes

Answer 10

Isolates DNA strands at by cleaving at sites
Cuts at palindromes to leave sticky or blunt ends
Used to see DNA
1. Separate by electrophoresis and fluoresce
2. Look at strands using audio radiography

Question 11

Hybridization and Probes

Answer 11

Detects specific nucleotide sequences
Need other techniques to actually see it
Homo (DNA/DNA) or hetero (DNA/ RNA) duplexes
Extreme conditions raise stringency, harder to hybridize
Shorter probes are harder to bind with

Question 12

Restriction Length Polymorphism

Answer 12

Used to look at differences in cleavage sites

1. Cut with enzymes
2. PCR
3. Southern blotting
4. Gel electrophoresis

Question 13

Southern Blotting

Answer 13

Looking for DNA through auto radiography

1. Restriction enzymes
2. Electrophoresis, blot
3. Hybridize with labeled cDNA
4. Autoradiography

Question 14

Southern Blotting

Answer 14

Look at RNA expression

1. Gel electrophoresis, blot
2. Hybridize with labeled cDNA
3. Visualized with UV probe over time

Question 15

DNA Microarray

Answer 15

Measure expression of many genes at once

1. Isolate mRNA
2. Reverse transcribe into DNA
3. Hybridize to probes in chip
4. Fluoresce

Question 16

Allele Specific Oligonucleotides

Answer 16

Detect mutations in alleles

1. Prepare chip with ASO’s
2. Wash with DNA solution
3. Blot colors will correspond with condition

Question 17

PCR

Answer 17

Try to amplify specific DNA sequence

1. Heat and separate strands
2. Cool and hybridize with primers
3. Polymerase
4. Repeat to isolate and amplify sequence of interest

Question 18

ASO PCR

Answer 18

Amplify a specific allele
Add primer for allele
TAQ won’t extend if it isn’t 100% attached

Question 19

Reverse Transcriptase (RT) PCR

Answer 19

Quantify mRNA in tissue

1. Convert mRNA to cDNA with RT
2. Amplify cDNA

Question 20

Sanger DNA Sequencing

Answer 20

Urea polyacrilimide gel electrophoresis
Use ddNTP’s
Read from bottom to top, so need to interpret from top to bottom

Question 21

Automated DNA Sequencing

Answer 21

PCR with ddNTP’s followed by capillary gel electrophoresis

Use laser detector to see sequence

Question 22

Automated DNA Sequencing

Answer 22

PCR with ddNTP’s followed by capillary gel electrophoresis

Use laser detector to see sequence

Question 23

SalI vs XhoI

Answer 23

Sal is GAC Xho is GAG

Question 24

[Salt] and Hybridization

Answer 24

Higher [salt] means better hybridization