Zaidi: Analyzing Cell, Molecules and Systems 1

Question 1

Primary cell culture

Answer 1

Derived directly from animal

Usually survive for a finite period of time

Question 2

Established or continuous cell lines

Answer 2

Primary culture that has been made immortal

Question 3

Sub-cellular fractionation

Answer 3

Reduces complexity of material

Question 4

Centrifugation

Answer 4

Separate different cell types based on size/density

Question 5

Ultracentrifugation

Answer 5

Separate organelles

Question 6

Lysis of synaptosomes gives

Answer 6

Synaptic plasma membrane

Question 7

What is enriched in synaptic plasma membrane

Answer 7

Calcium and ATPase

Question 8

Recombinant DNA technology use

Answer 8

Used to overexpress a protein to purify it more easily

Question 9

PMCA activity inhibited by

Answer 9

Reactive oxygen species

Question 10

Techniques used to analyze proteins

Answer 10

SDS-PAGE
Western blotting
ELISA
Mass spectrometry

Question 11

SDS-PAGE used to

Answer 11

Analyze unkown proteins

Can visualize separated proteins with stains/dyes

Question 12

SDS is

Answer 12

Largely hydrophobic with a negative charge

• Unfold proteins
• Gives all proteins uniform charge

Question 13

Beta-mercaptoethanol

Answer 13

Present in SDS-PAGE

Reduces disulfides/further denatures protein

Question 14

Western blotting/immunoblotting

Answer 14

Analyzing specific/known proteins

Question 15

Western blotting technique

Answer 15

Resolve protein samples on PAGE
Transfer fractionated proteins from gel onto membrane
Block membrane with neutral protein
Incubate membrane with HRP-labeled antibody
Incubate blot with chemiluminescent HRP substrate and expose to film

Question 16

Western blotting antibody explanation

Answer 16

Introduce primary antibody to antigen in question

Introduce secondary antibody with markers

Question 17

ELISA

Answer 17

Tests for the levels of specific antigen or antibody concentrations using corresponding antigen or antibody

Question 18

ELISA simple breakdown

Answer 18

Probe patient serum with either test antibody or antigen
Antigen/antibody is conjugated to reactive (color-changing) enzyme
Antigen/antibody presents with color change

Question 19

Indirect ELISA purpose

Answer 19

Measure the amount of an antibody in a samle

Question 20

Sandwich ELISA purpose

Answer 20

Measure the amount of an antigen in a sample

Question 21

Indirect ELISA process

Answer 21

Start with antigen coated well
Specific antibody binds antigen
Enzyme-linked antibody added-binds to specific antibody
Substrate is added, enzyme causes color change, rate of color change is proportional to amount of specific antibody

Question 22

Sandwich ELISA process

Answer 22

Monoclonal antibody coated well
Antigen binds to antibody
Second monoclonal antibody w/enzyme binds to immobilized antigen
Substrate added, enzyme causes color change, rate of color change is proportional to amount of antigen

Question 23

Mass spectrometry used to

Answer 23

Identify unknown proteins

Question 24

Mass spectrometry requires

Answer 24

Tryptic digestion products (peptide fragments)
Ionization (charge)
Detection method
Computer databases with known protein sizes