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Flashcards in Tools of Protein Biochemistry Deck (73):
1

What are two major types of electrophoresis

1.) Native-PAGE (non-denaturing)
2.) SDS-PAGE (denaturing)

2

Separates molecules according to their charge, shape, and size

Electrophoresis

3

How many proteins are expressed in our bodies?

20,000

4

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

Net charge

5

Electrophoresis requires a gel for

Support and Molecular sieving

6

What does PAGE mean in SDS-PAGE?

Polyacrylimide gel electrophoresis

7

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

-Non-denaturing

Native-PAGE

8

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

Native PAGE

9

In native PAGE, the migration of proteins depends on

Size, shape, and native charge

10

Single amino acid substitution of Glutamate 6 with valine

-causes conformational change that affects hemoglobins ability to bind oxygen

Sickle Cell Disease

11

Hemoglobin A is a tetramer composed of what 4 subunits?

2α and 2β

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

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

13

Which Hb migrates faster during electrophoresis at pH 7?

HbA (it is negatively charged)

14

Which Hb migrates faster during electrophoresis at pH 4?

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

15

Electrophoresis method to separate proteins baed on molecular weight

SDS-Page

16

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

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

2.) SDS

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

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

Sodium dodecylsulfate (SDS)

18

What fragments moves faster in SDS-PAGE?

Small fragments

19

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

Logarithmic

20

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

SDS-PAGE

21

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

Erythropoietin

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

Hemoglobin production

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

Red blood cells by bone marrow

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?

Western Blot

25

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

Size

26

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

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

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Portion of a molecule to which an antibody binds

Epitope

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The antigen binding site of an antibody

Paratope

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Epitopes can be composed of

Sugars, lipids, or amino acids

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

Indicator

31

What are two common indicators?

Alkaline phosphatase and Horseradish peroxidase

32

Summarize a western blot

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.

33

Combine electrophoresis and immunological techniques

Western Blots

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

Enzyme-Linked Immunoabsorbant Assay (ELISA)

35

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

ELISA

36

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

ELISA

37

Which is more specific, Western Blotting, or ELISA?

Western Blotting

38

Explain how ELISA works

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.

39

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

Direct ELISA

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?

Indirect ELISA

41

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

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

42

ELISA is more sensitive than a western blot because?

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)

43

What is the gold standard for HIV testing?

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

44

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

Window Period (4-6 weeks)

45

What indicates a positive HIV Western Blot?

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

46

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

Indeterminate

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What are some causes of an indeterminate HIV western blot?

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

48

What is the procedure following an indeterminte result?

Retest in > 6 weeks

49

What does the Western blot HIV test actually measure?

The antibody to HIV

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What are the antibodies to HIV bound to?

Antigen to HIV

51

Inadequate blood supply to an organ or part of the body

-Cause of myocardial infarction

Ischemia

52

Serve as useful diagnostic markers of acute ischemia

Troponin, Myoglobin, and Creatine Kinase

53

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

Myocardial infarction

54

The contractile regulating protein of striated muscle

Troponin

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

Acute Myocardial Infarction

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

Four days following the event

57

What is the difference between HIV ELISA and Troponin ELISA?

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

58

Describe how co-immunoprecipitation works

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

59

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

Loss of p53

60

p53 tumore suppressor is frequently disabled by

Mutations

61

Wild type in 50% of human cancer

p53 gene

62

In response to cellular stress, promotes p53 ubiquination and degredation by the proteasome

-An E3 ubiquitin ligase

MDM2 (HDM2)

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Targets p53 for degredation to disable the p53

Overexpression of MDM2 in human cancer

64

How did scientists show that MDM2 and p53 interact?

Immunoprecipitation of MDM2 which showed coprecipitation of p53

65

Describe immunoaffinity Chromatography

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

66

Purifying proteins from cell extracts

Affinity Purification

67

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

Yes

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

Immunohistochemistry

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

HER2-positive breast cancer

70

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

HER2

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Tend to be more aggresive than other types of breast cancer

HER2-positive breast cancers

72

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

Epitope tag

73

How does epitope tagging work?

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

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