Tools of Protein Biochemistry

Question 1

What are two major types of electrophoresis

Answer 1

1. ) Native-PAGE (non-denaturing)

2. ) SDS-PAGE (denaturing)

Question 2

Separates molecules according to their charge, shape, and size

Answer 2

Electrophoresis

Question 3

How many proteins are expressed in our bodies?

Answer 3

20,000

Question 4

Buffer pH and intrinsic chemical properties of macromolecules (DNA, RNA, and proteins) determine their

Answer 4

Net charge

Question 5

Electrophoresis requires a gel for

Answer 5

Support and Molecular sieving

Question 6

What does PAGE mean in SDS-PAGE?

Answer 6

Polyacrylimide gel electrophoresis

Question 7

Electrophoresis where the protein retains it’s activity throughout the process

-Non-denaturing

Answer 7

Native-PAGE

Question 8

Polypeptides retain their higher-order structure and often retain enzymatic activity and interaction with other polypeptides in

Answer 8

Native PAGE

Question 9

In native PAGE, the migration of proteins depends on

Answer 9

Size, shape, and native charge

Question 10

Single amino acid substitution of Glutamate 6 with valine

-causes conformational change that affects hemoglobins ability to bind oxygen

Answer 10

Sickle Cell Disease

Question 11

Hemoglobin A is a tetramer composed of what 4 subunits?

Answer 11

2α and 2β

Question 12

What significance does the fact that a glutimate (negative charge) is substituted for a valine (neutral charge) in people with sickle cell?

Answer 12

HbA and Hb-S can be separated at physiological pH because HbA is negatively charged

Question 13

Which Hb migrates faster during electrophoresis at pH 7?

Answer 13

HbA (it is negatively charged)

Question 14

Which Hb migrates faster during electrophoresis at pH 4?

Answer 14

HbA and Hb-S have the same charge and thus can not be separated

Question 15

Electrophoresis method to separate proteins baed on molecular weight

Answer 15

SDS-Page

Question 16

Prior to SDS-PAGE, proteins are treated with which two things?

Answer 16

1. ) Reducing agent to break disulfide bonds (β -mercapthanol)
2. ) SDS

Question 17

An ionic detergent that denatures secondary and non-disulfide-linked tertiary structures

-Binds to protein backbone and confers a negative charge proportional to mass

Answer 17

Sodium dodecylsulfate (SDS)

Question 18

What fragments moves faster in SDS-PAGE?

Answer 18

Small fragments

Question 19

The movement in SDS-PAGE is not linear, but rather

Answer 19

Logarithmic

Question 20

Cyclists, distance runners and crosscountry skiers have been utilizing recombinant human erythropoietin (RhEPO) to improve their endurance performance. How can we detect Erythropoietin?

Answer 20

SDS-PAGE

Question 21

Induces hemoglobin production which allows more oxygen to be carried in the blood and improves endurance

Answer 21

Erythropoietin

Question 22

Erythropoietin was created for people with anemia or people undergoing chemo to help increase

Answer 22

Hemoglobin production

Question 23

Erythropoietin (EPO) is a hormone produced by the kidney that promotes the formation of

Answer 23

Red blood cells by bone marrow

Question 24

Blood does not have many circulating proteins in it, but in the cell their are many. How can we isolate a specific protein of interest?

Answer 24

Western Blot

Question 25

In all types of blotting,we make our molecules negatively charged so that we are separating exclusively by

Answer 25

Size

Question 26

How do we generate antibodies that bind our target protein in a western blot?

Answer 26

Inject a rabbit with the antigen (target protein). Take serum from the rabbit. The supernatant will contain the desired antibodies

Question 27

Portion of a molecule to which an antibody binds

Answer 27

Epitope

Question 28

The antigen binding site of an antibody

Answer 28

Paratope

Question 29

Epitopes can be composed of

Answer 29

Sugars, lipids, or amino acids

Question 30

Antibody with a bound enzyme that can catalyze | conversion of a colorless molecule to a colored one

Answer 30

Indicator

Question 31

What are two common indicators?

Answer 31

Alkaline phosphatase and Horseradish peroxidase

Question 32

Summarize a western blot

Answer 32

The antibodies specific to the protein of interest are generated by injecting a rabbit with the antigen (protein of interest) and taking blood serum from the rabbit. Next, gel electrophoresis is performed on the sample containing our target protein. Upon completion, the wells are transferred from the gel to a nitrocellulose sheet. The antibodies we generated are added to the sheet, at which point they bind the target protein. All other antibodies are washed way and tagged antibody is added (indicator), which allows us to isolate the desired protein.

Question 33

Combine electrophoresis and immunological techniques

Answer 33

Western Blots

Question 34

First thing you do when screening for a disease. A faster, more sensitive method than western blotting to detect antibodies or antigens in a patient

Answer 34

Enzyme-Linked Immunoabsorbant Assay (ELISA)

Question 35

Antibodies developed by the human body against AIDS can be detected by

Answer 35

ELISA

Question 36

Troponin, released after a myocardial infarction, can be detected by

Answer 36

ELISA

Question 37

Which is more specific, Western Blotting, or ELISA?

Answer 37

Western Blotting

Question 38

Explain how ELISA works

Answer 38

You have a well, to which you can link an antigen or an antibody, depending on which one you are testing for. Next, add an enzyme antibody, which will bind to the antigen and allow for detection.

Question 39

Tagged antibodies bind directly to the antigen in the well and allow for detection in?

Answer 39

Direct ELISA

Question 40

A primary antibody binds directly to the antigen in the well. Then, tagged secondary antibodies bind to the primary antibody and allow for detection in?

Answer 40

Indirect ELISA

Question 41

How is the antigen actually detected in a western blot or ELISA?

Answer 41

A substrate is added that binds the enzyme attached to the antibody and produces either a fluorescent or colored product

Question 42

ELISA is more sensitive than a western blot because?

Answer 42

The well is hooked up to a spectrophotometric detector. Where as in a western, we have to observe the color change with our eyes (requires a larger sample)

Question 43

What is the gold standard for HIV testing?

Answer 43

Perform an ELISA, if it' negative than the patient is negative. If it is positive, perform a Western blot for confirmation. The blot results will provide the official diagnosis

Question 44

The time between HIV infection and when antibodies to HIV are produced

Answer 44

Window Period (4-6 weeks)

Question 45

What indicates a positive HIV Western Blot?

Answer 45

You need one band to be present in the region of gp 160 or gp 120, and one band present in the region of p31 or p24

Question 46

When bands are present but the pattern does not meet the criteria for positivity, the result is

Answer 46

Indeterminate

Question 47

What are some causes of an indeterminate HIV western blot?

Answer 47

Recent infection, advanced HIV, certain strands of HIV, etc.

Question 48

What is the procedure following an indeterminte result?

Answer 48

Retest in > 6 weeks

Question 49

What does the Western blot HIV test actually measure?

Answer 49

The antibody to HIV

Question 50

What are the antibodies to HIV bound to?

Answer 50

Antigen to HIV

Question 51

Inadequate blood supply to an organ or part of the body -Cause of myocardial infarction

Answer 51

Ischemia

Question 52

Serve as useful diagnostic markers of acute ischemia

Answer 52

Troponin, Myoglobin, and Creatine Kinase

Question 53

Cardiac cells rupture and release proteins into the blood. As a consequence, the levels of Troponin T increase, which indicates

Answer 53

Myocardial infarction

Question 54

The contractile regulating protein of striated muscle

Answer 54

Troponin

Question 55

The cardiac isoforms of troponin T and I increase in serum after

Answer 55

Acute Myocardial Infarction

Question 56

Troponin is highly specific for myocardial infarction because it is not elevated in any other trauma, heavy exercise, or renal failure. Also, it remains in the blood for up to

Answer 56

Four days following the event

Question 57

What is the difference between HIV ELISA and Troponin ELISA?

Answer 57

Troponin ELISA uses an immobilized antibody in the well. Where as HIV used an immobilized antigen

Question 58

Describe how co-immunoprecipitation works

Answer 58

We bind beads to an antibody specific to our target protein and add it to our sample. The antibody binds the target protein.The increased mass from the beads causes the complex to precipitate out. If our target protein is complexed with another protein, that protein will also precipitate out. We can separate the two by SDS page and then perform a western blot

Question 59

Allows the proliferation of damaged cells, which can lead to tumor development

Answer 59

Loss of p53

Question 60

p53 tumore suppressor is frequently disabled by

Answer 60

Mutations

Question 61

Wild type in 50% of human cancer

Answer 61

p53 gene

Question 62

In response to cellular stress, promotes p53 ubiquination and degredation by the proteasome -An E3 ubiquitin ligase

Answer 62

MDM2 (HDM2)

Question 63

Targets p53 for degredation to disable the p53

Answer 63

Overexpression of MDM2 in human cancer

Question 64

How did scientists show that MDM2 and p53 interact?

Answer 64

Immunoprecipitation of MDM2 which showed coprecipitation of p53

Question 65

Describe immunoaffinity Chromatography

Answer 65

We add antibodies specific to our target protein to a column. We then add a cell extract to the column. As the cell extract moves through the column, the target protein will bind the antibodies and everything else will be eluted. Then we prepare a solution to elute our target protein, which we can then load to the gel and quantify

Question 66

Purifying proteins from cell extracts

Answer 66

Affinity Purification

Question 67

Can immunoaffinity chromatography be used for interaction proteins similar to how co-immunoprecipitation worked?

Answer 67

Yes

Question 68

Uses primary antibodies to target and label specific proteins in tissue and then secondary antibodies with a conjugated enzyme to bind the primary antibodies -Key is that it is in the tissue

Answer 68

Immunohistochemistry

Question 69

A breast cancer that tests positive for a protein called human epidermal growth factor receptor 2 (HER2), which promotes the growth of cancer cells

Answer 69

HER2-positive breast cancer

Question 70

In about 1 out of every 5 breast cancers, there is a gene mutation that results in the roduction of excess

Answer 70

HER2

Question 71

Tend to be more aggresive than other types of breast cancer

Answer 71

HER2-positive breast cancers

Question 72

Added to molecules that an investigator wants to visualize when we can't develop an antibody for our protein of interest

Answer 72

Epitope tag

Question 73

How does epitope tagging work?

Answer 73

We make and add a tag to the DNA sequence of the desired molecule, which results in the expressed protein being made with the tag in its amino acd sequence. We can then generate an antibody specific to the tag and detect with a blot