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How does protein concentration vary across cells?

No cell contains all proteins possible coded for by the DNA. Some proteins are similar so small or large differences can separate them

1

Setting the proteins free

Break up the cells (lyse them) by different techniques

Sonication, homogenization, osmotic pressure

2

Sonication

Room with sonicator (loud room) put sample in sonicator and its blasted with sound waves and cell membranes lyse

3

Homogenization

Metal ball bearing, machine that shakes it that breaks up cell membranes

4

Osmotic pressure

Salt, put in water and the membranes blow up

5

Procedures of homogenation

Break cells with high frequency sound

Use a mild detergent to make holes in the plasma membrane

Force cells through a small hole using high pressure

Shear cells between a close fitting rotating plunger and thick walls of a glass vessel

6

What is in the homogenate?

Contains large and small molecules from the cytosol such as enzymes, ribosomes, and metabolites, as well as the membrane bound organelles

Wen done carefully most of the membrane bound organelles are left in tact

7

Taking the cells for a spin (types of centrifugation)

•  Differential Centrifugation

•  Rate Zonal Centrifugation

•  Equilibrium Density Gradients

8

Sedimentation rate equation

s = m[1-(do/d)]/f

9

Differential centrifugation

•  Differential centrifugation separates matters according to mass and density.

•  Can be used to separate organelles and large protein complexes- good for purifying out big things

10

Process of differential centrifugation

Homogenate filtered many times and then poured it to leave highest density items in previous tubes

Nuclei/ mitochondria, chloroplasts, lysosomes, peroxisomes/ plasma membrane, large polyribosomes, ER fragments/ ribosomal subunits, small polyribosomes/ soluble portion of cytosol(including proteins)

11

Rate-zonal centrifugation

Sucrose gradient is used to provide density stability during centrifugation

Particles of a similar density remain in the fraction because of mass and shape

Better resolution of separation

You have substances of known density and you separate cell particles by how they fall in this scale. Separate and study fractions

12

What happens after fractionation?

•  What is actually in each fraction collected during centrifugation?

•  Utilize column chromatography

13

Column Chromatography

•  Separate mixture ofproteins based on different properties of the protein

•  Size
•  Charge
•  Binding affinity

14

Gel filtration

Size exclusion chromatography

"Matrix"= stationary beads in column, small proteins trapped by pores in the beads and the flow more slowly than the large proteins that flow around the beads

Large proteins appear in the earlier fractions

May have to do more than once

15

Ion exchange chromatography

Cation (negatively charged beads) and anion (positively charged beads) columns

Proteins move through columns at rates determined by their net charge at the pH being used

16

How do you get the proteins out of the ion column?

Elute charged protein with solution (ex: if negatively charge protein, use salt solution)

The added solution outcompetes for the charge and binds to the beads, causing the proteins to come off

17

Affinity chromatography

Protein mixture is added to column containing a polymer-bound ligand specific for protein of interest

Results in highly specific purification, covalent bonds

Use a competing molecule, salt, or change in pH to remove the protein from the column

18

Which fraction has the protein of interest?

Assay to determine presence of protein in the fraction

19

Protein Purification

•  Purification techniques to isolate a specific protein from all other cellular components

  Basic steps in protein analysis:
•  Protein assays (why start with an assay?)
•  Protein purification
•  Look for specific activity- make sure you purified just your protein

20

Biochemical assay

Measure how much reactants and products you have

Give substrates and see if products are produced

21

Ways to detect proteins

•  Optical density: Concentration of protein can be evaluated

•  Gel electrophoresis

•  Immunological methods (antibodies)

•  Biochemical Assays

22

What various properties of a protein can be examined with gel electrophoresis?

Monomeric proteins: single subunit

Multimeric proteins: more than one subunit

Homodimers: dimeric proteins where both subunits are identical.

Heterodimers: dimeric proteins where both subunits aredifferent.

23

SDS Page

SDS polyacrylamide gel electrophoresis

Proteins are treated first with SDS and β-mercaptaethanol

SDS maintains the denatured conformation of proteins and coats the polypeptides with a negative charge, have an equivalent charge/mass ratio

β -mercaptoethanol reduces disulfide bonds

24

Process of SDS Page

Denature and coat with SDS

Place mixture of proteins on gel, apply electric field

Stain to visualize separated bands

25

How do you use protein specific stains?

You can detect the polypeptides in the gel

First you run the gel just to see if the protein is present, then you purify more and run the gel again. (Can't use affinity column right away because if there is a lot of stuff it doesn't work as well)

26

2-D Gel Electrophoresis

Protein samples containing a large number of proteins can be separated using electrophoresis in two dimensions

The first dimension is through a gel containing a pH gradient. Proteins migrate until they reach the pH at which they have no charge.

The second dimension is generally an SDS gel, which separates according to size

27

Separation in the first degree of 2D electrophoresis

Separation in the first dimension is based on isoelectric point (net 0 charge)of a protein. When it reaches this point, the protein stops migrating

•  Charge on a protein is sum of all charges on amino acid side chains
•  As the pH goes from low to high (4-10), amino acids change their ionization state

28

Where are the positive and negative electrodes in 2D gel electrophoresis

Electrophoresis in the first gel has the positive electrode on top and the negative electrode on the bottom

The reverse is true for electrophoresis through the SDS gel.

29

What makes proteins move according to pH?

high hydrogen content at low pH (acidic), + charges move to the basic side (high pH)

30

What is surprising about 2D

Can resolve more around 2000 proteins at once

2D gels can resolve proteins thatdiffer by a single unit of charge

31

Western blot

Can help you find one specific protein

Uses antibodies

32

Process of western blotting

Run an electric current through an SDS gel onto a nitrocellulose filter that binds to the proteins

Incubate with an antibody for that protein, then wash

Incubate with secondary chromagenic antibody that binds to the first antibodies (alkaline phosphatase)

Substrate is added that the secondary antibody cleaves and color shows where the protein is on the gel

33

ELISA

Cell culture plate

Well is coated with antigen

Add plasma from test individual. If antibody is present it binds to antigen

Secondary antibody binds to antibody in plasma (Alkaline phosphatase)

Test enzyme catalyzes a reaction that changes the color of the solution indicating presence of antibody

34

Home pregnancy tests

Antibody to Chorionic Gonadotropin on strip

Does urine contain CG?

Enzyme linked secondary antibody to CG that gives color reaction

35

immunofluorescence

Can detect proteins in cell

Kinesin (yellow)
Tubulin (green)
Overlap (orange)

Useful in cell biology to see where things actually occur in a cell and their arrangement

36

Edman degradation

•  This technique allows sequential analysis of amino acids starting at the N-terminus

•  Approximately 30 or so amino acids can be clearly determined.

•  To sequence an entire protein, small sections have to be determined and then pieced together (constant washing with acid not good)

37

Process of Edman degradation

Phenylisothiocyante (PITC)- Binds to amino end of protein, label that first amino acid. Specific acid that recognizes pitc and it cleaves bind between first and second amino acid

Now you have a lot of the first amino acid in the solution, repeat.

38

Proteins can be broken into smaller peptides for sequence determination

•  Start with a purified protein

•  You can use proteases that cleave bonds between specific amino acid pairs

• These can then be subjected to Edman degradation.

•  The entire protein sequence can be pieced together only after sequencing of two sets of protease fragments.

What order were they in? Cut different parts with second protease, line up AA by looking at overlapping fragments

39

Proteomics

•  Study of all proteins produced in cell or species under a givenset of conditions.

•  2D gel electrophoresis coupled with mass spectrometry to identify MW of protein