Lecture 3 Slides Purifying And Analyzing Proteins Flashcards Preview

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1

What does protein kinase phosphorylate

The hydroxyl group of a serine, threonine, or tyrosine

2

What does phosphorylation affect

Conformation of a protein and interactions with ligands

3

Histidine kinases

Signaling proteins found in prokaryotes, fungi, and plants

4

Protein Phosphatases

Perform reverse of protein kinase
Less specific than protein kinase with regard to substrate
Just as important for controlling activity of the protein targets

5

Can a protein be a target for multiple kinases

Yes. Each kinase would target a different amino residue

6

Cell fractionation

Centrifugation to separate cell components based on size, mass, density

7

Goals of using cell fractionation

Enrichment of organelles for protein purification
Cell free systems to study motility, enzymatic reactions, etc.

8

Fractionation methods 4

Break cell with osmotic shock, mechanical force, nonionic detergent
Plasma membrane and other membrane systems form vesicles
May need to include inhibitors of proteases, reducing agents
Some organelles remain intact

9

Cytoplasm

Contents of eukaryotic cell outside of nucleus

10

Cytosol

Contents of cytoplasm outside of membrane-bound organelles

11

How does cell fractionation by differential (rate-zonal) centrifugation work

Separation is based on size, density of organelles or large protein complexes
Cell homogenate is placed in tube
Slow centrifugation
Pellet contains whole cells, nuclei, cytoskeletons
Subject supernatant to medium speed centrifugation
Pellet contains mitochondria, lysosomes, peroxisomes
Supernatant subjected to high speed centrifugation
At cell free system, can use this supernatant for in Vitro translation
Pellet contains microsomes and small vesicles
Supernatant subjected to very high speed centrifugation
Pellet contains ribosomes, viruses, large macromolecules

12

Microsomes

Vesicles produced by fragmentation of endoplasmic reticulum when cell is organized

13

Velocity sedimentation

Particles are separated by size and shape; a density gradient prevents mixing. The gradient is formed prior to centrifugation.

14

S-value

Sedimentation coefficient - depends on Mass, volume, surface area of particle

15

Stages of velocity sedimentation

Place multi sediment sample in tube over stabilizing sucrose gradient

Centrifugation
A layer of fast sedimenting component forms under slow sedimenting layer. There is sucrose above, below, and in between layers

Fractionation

16

Example of velocity sedimentation. What do results mean?

Example of ribosomes and polyribosomes. You see 40s and 60s, and then 80s. You know this is a eukaryotes because of the 40 and 60. The polyribosome made by those two parts will be less dense, at 80, than th sum of their individual densities

17

Equilibrium density gradient sedimentation

Density gradient is formed mechanically (sucrose gradient) or by centrifugation (cesium chloride gradient); particles migrate to a point in the density gradient where they are in density equilibrium with th surrounding solvent.

18

Density gradient mechanical

Sucrose gradient

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Density gradient by centrifugation

Cesium chloride gradient

20

In a sucrose gradient, in what order do fractions line up

Bottom to top, high buoyant density to low buoyant density

21

If you separate dna from RNA, which has higher density

RNA

22

How can you density separate different DNAs

By different base compositions (GC rich vs AT rich)

23

Column chromatography

Based on charge and size
An anion exchange column has positively charged matrix that interacts with negative charges on the proteins. Positively charged matrix will slow down negative particles as it interacts with them.

24

Two types of chromatography

Ion-exchange
Gel filtration

25

Gel filtration chromatography

Matrix of porous beads will trap and retard smaller molecules that can get caught in it, thus separating small from large molecules

26

What is ionic buffer

It is a charge that reacts with an opposite to slow it down

27

Affinity chromatography

Brads with covalently attached substrates bind specific molecule letting the rest pass

28

What can be bound to beads in affinity chromatography

Substrate for enzyme
Antibody
Ligand
-peptide, DNA fragments, metal ion (Ni, Cu)

29

How does eluting solution separate what is bound to beads in affinity chromatography

Eluting solution will contain component that decreases binding. For example, a binding competitor

30

SDS-PAGE

Sodium dodecyl sulfate pokyacrylamide gel electrophoresis

31

How does SDS PAGE work

Denature proteins by boiling them in SDS
Heat denatures proteins
Ionic detergent binds hydrophobic regions on proteins
Coats proteins with negative charge relative to size of protein

Beta-mercaptoethanol
Reducing agent breaks disulfide bonds within and between proteins

32

How does SDS gel electrophoresis work

Gel is in plastic casing.
Top has negative cathode,
Bottom has positive anode.
Ample loaded onto gel by pipette.
Choose, for example, two samples. One with two proteins joined by a disulfide bond and the other a single protein.
Heat with SDS and mercaptoethanol.
Protein complex splits into two proteins covered with electrons. Single protein does not break but is covered in negative charges.
The larger particles will bind more to gel and go down more slowly than smaller particles.

33

Two dimensional gel electrophoresis

First dimension separates proteins based on isoelectric point in a pH gradient net (proteins treated with reducing agent but no SDS!)
Second dimension, separates proteins by molecular weight - first dimension strip is subjected to SDS-PAGE

34

How do you separate proteins by isoelectric point

At isoelectric point, the protein has no net charge and therefore no longer migrates in the electric field. So, suppose a protein is positively charged, low pH. It will increase in pH until it reaches its isoelectric point. Sam for high pH protein but in opposite direction.

35

Why do isoelectric point to separate peotein by molecular weight.

Because they will be neutral and will both only differ by weight when in gel

36

What does phosphorylation change?

It results in a change in the isoelectric point of a protein.

37

How does phosphorylation happen

Protein kinases transfer a phosphate group from ATP to a target protein

38

What are targets for phosphorylation

Hydroxyl groups on:
Serine
Threonine
Tyrosine

39

How does mass spectrometry work

MS is used for protein identification
A single protein spot is excised from gel
Peptides are released by tryptic digestion and their masses measured using maldi-tof mass spectrometry
Graphs m/z(mass to charge ratio) to abundance (on y axis)
Protein sequence databases searched for matches with theoretical masses calculated for all trypsin related peptides

Gene is isolated and identified

40

How does MS-MS work

Mass spectrometer gives peptide masses
Each peptide is then further fragmented at peptide bonds
Masses of fragments measured on a coupled second mass spectrometer
The mass differences bet. Fragments can be used to construct a partial amino acid sequence. The data may allow gene ID, or provide the means for cloning the gene.
Post translational modifications can be detected

41

Proteomics

Identification of a collection of proteins expressed
-in a specific cell type
-in a specific organelle
-under a specific set of environmental conditions
-in response to disease
-identifies post translational modifications such as phosphorylation
-relative concentrations of proteins can be compared

42

Proteomics methods

-protein fragmented by trypsin digestion; peptides separated by liquid chromatography
-MS-MS of peptide fractions
-comparison of peptide sequences to predicted proteins from genome sequence

43

Two examples of clinical proteomics

-blood or cerebral spinal fluid, or urine samples are subjected to protein fractionation and MS-MS analysis
-"bio markers" - proteins associated with disease state are discovered by comparing samples from normal and disease samples

44

Where are antigen binding sites on antibodies

NH2 ends of light and heavy chains

45

Epitope

Antigenic determinant. Consists of 5 to 7 amino acids or 1-6 monosaccharides

46

How is strength of antibody binding defined

By affinity or association constant
(Inverse of dissociation constant)
Ka = [AgAb]/[Ag][Ab]

47

What is true at concentration of free antigen required to fill half the sites on the antibody

[AgAb]=[Ab]
Ka = 1/[Ag]
Units are liters per mole

48

Where are the hyper variable regions of light and heavy chains

Inside the binding site

49

Where are the variable regions of light and heavy chains

Outside of binding sites

50

How is antibody diversity generated

Somatic cell combination occurs during B lymphocyte development in the bone marrow

51

How does recombination happen for antibodies

VDJ recombinase selects specific V, D, J regions. Imperfect joining creates additional variability.

52

What kind of diversity exists in human antibodies

> 10^12 different antibodies

53

Is each antibody unique

Each B cell produces a unique antibody as a result of the recombination

54

Are there different types of immunoglobulins (antibodies)

Yes, depending on which C region is found on the heavy chain

55

Process to produce antibodies

1. Purify protein of interest to homogeneity
2. Inject antigen into animal
3. Immune response - if the antigen binds to the body on a B cell, that cell is activated
4. Test serum over several weeks for presence of specific antibody
5. Recover antibodies from serum (polyclonal antibody)

56

How does immune response happen

1. A naive cell, B lymphocyte is first exposed to antigen (in bone marrow)
2. The cell creates memory cells and effector cells. More of the latter.
3. The memory cells act during a second exposure to the antigen, and there is an even stronger response, with even more memory cells and effectors

57

Polyclonal antibody

Consists of many different antibodies, each produced by a different clone of B cells and each recognizing and binding to an epitope.

58

How many epitope a may a single antigen have

Many

59

How many epitopes will a polyclonal antibody recognize on an antigen

Many

60

Monoclonal antibody

Derived from a clone of cells from a single B cell producing a single type of antibody molecule

61

How is a monoclonal antibody produced

1. A mouse is immunized with antigen X and a mutant cell line is derived from a tumor of B lymphocytes
2. The mouse creates antibody.
3. The mouse B lymphocytes die a few days after culture. The mutant cells grow indefinitely in a normal medium.
4. Dead lymphocytes and living cells are fused.
5. Fusion products played in multiple wells
6. Only hybridomas grow on the selective medium
7. Test supernatant for anti-X antibody and redistribute cells from positive well at ~1 cell per well.
8. Allow cells to multiply then test supernatant for antibodies
Positive clons will provide a continuous source of antibodies

62

How many epitopes will a monoclonal antibody recognize

One

63

Multi silent antigen

Has multiple epitopes

64

Western blotting or immunoblotting process

Proteins from gel transferred to a membrane
Membrane is incubated with primary antibody against protein of interest
Unbound antibodies washed away
Membrane incubated with enzyme-linked secondary antibody against primary antibody
Unbound antibodies washed away
Product of enzyme reveals protein(s) bound by primary antibody

65

How are secondary antibodies marked

With colorimetric or chemiluminescent marker

66

Why use secondary antibody

It amplifies the signal and is conveniently available from a commercial source.

67

How are protein kinases used

Control activity and stability of target proteins
Regulate protein protein interactions
Signaling pathways