Proteins (Isolation Techniques).pdf

7/22/2019 Proteins (Isolation Techniques).pdf

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CELL AND MOLECULAR BIOLOGY

Prepared by: Jassy Mary S. Lazarte & Jeremy G. Vicencio

Department of Biology, University of the Philippines ManilaBio150 Lab

PROTEINS


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What are Proteins?

Protein from the Greek proteios (primary, first, and

foremost)

Proteins are the work horses of biological systems.

They play key roles in constructing and maintaining livingcells

Our genes code for proteins

Proteins are polymers of amino acids


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Proteins are

Polypeptides + (cofactors, coenzymes, prosthetic

groups, other modifications)

Polypeptides are covalently linked -amino acids

Cofactors are non-amino acid components e.g. metal ionslike Zn2+ in carboxypeptidase

Coenzymes are organic cofactors e.g. nucleotides in lactate

dehydrogenase

Prosthetic groups are covalently attached cofactors e.g.heme in myoglobin


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Proteins and their roles

Enzymes (biological catalysts)

Hormones

Storage proteins

Transport proteins

Structural proteins

Protective proteins

Contractile proteins

Toxic proteins


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Proteins and their structure


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How to isolate proteins?

1. Lyse the cell

2. Solubilize the proteins

use detergents in the protein extraction buffer

a. Nonionic detergents (milder)

ex. Triton X-100: break lipid-lipid interaction and

lipid-protein interaction

b. Anionic detergents (more denaturing)ex. SDS: protein-protein interaction,Sodium Deoxycholate: protein-protein interaction

*Upon lysis of the cell, proteases are released into the

lysate


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Protein Isolation

What are proteases?

aka proteinases, peptidases or proteolytic enzymes

are enzymes that break peptide bonds between amino

acids of proteins Where do proteases come from?

Animal cells: Lysosomes, contain a large variety of hydrolytic

enzymes that degrade proteins and other substances

Plant cells: Vacuole, many hydrolytic enzymes found invacuole resemble those present in Lysosomes of animal cells

other organelles also have proteases


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Protein Isolation

How to prevent protein degradation by

proteases

the protein isolation is carried out at low

temperature to minimize the activities of theseprotease

to further optimize the results, use the proteases

inhibitors


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Protein Isolation

3. Addition of Protease Inhibitors

EDTA (or EGTA): chelating the Ca2+

PMSF: a general serine protease inhibitor.

*It is the most common inhibitor used in protein purification.

*Soluble in isopropanol.

4. Quantify Protein


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Protein Quantitation

A. Colorimetric methods:

1. Biuret

2. Lowry

3. BradfordB. UV absorption method

*The amino acids tryptophan, tyrosine and phenylalanine absorb

light in the UV wavelength

Since the absorption is proportional to concentration, this is a

useful way to quantitate protein concentration (for proteins

containing Trp)


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Disadvantages of UV absorption method

If some proteins do not contain these amino acids, it will

not absorb UV light

Nucleic acids (DNA, RNA) contaminant will also absorbUV light


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A260/A280 has high

sensitivity for nucleic acid

contamination in protein:

A260/A280 lacks sensitivity

for protein contamination

in nucleic acids:

% protein % NA A260/A280

100 0 0.57

95 5 1.0690 10 1.32

70 30 1.73

% NA % protein A260/A280

100 0 2.00

95 5 1.9990 10 1.98

70 30 1.94


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Colorimetric methods

protein samples can be modified with appropriate

reagents to produce a color reaction and measure

protein concentration using a spectrophotometer.

Advantages of Colorimetric methods1. Cheap cuvette! (cheap glass or plastic versus

quartz)2. Not contaminating absorbance from nucleic acids!


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BRADFORD ASSAY

Bradford Method

A dye known as CBBG, Coomassie

Brilliant Blue G-250 was developed

by the textile industry. It was noticed to stain skin as well as

the textiles.

This dye (which normally absorbs at465nm) binds to proteins and to

absorb strongly at 595nm.

The assay is sensitive, but somewhat

non-linear


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Dye-Binding ( Bradford ) Assay

Bradford, MM. A rapid and sensitive for the quantitation of microgram

quantitites of protein utilizing the principle of protein-dye binding.AnalyticalBiochemistry 72:248-254. 1976.

Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182:50-69(1990).

CBBG primarily responds toarginine residues (eight times as

much as the other listed residues)

If you have an arginine rich protein,

You may need to find a standard

that is arginine rich as well.

CBBG binds to these residues in theanionic form

Absorbance maximum at 595 nm(blue)

The free dye in solution is in thecationic form,

Absorbance maximum at 470 nm

(red).


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Advantages Fast and inexpensiveHighly specific for protein

Very sensitive [1-20 g (micro assay) 20-200 g(macro assay)]

Compatible with a wide range of substances Extinction co-efficient for the dye-protein complex is

stable over 10 orders of magnitude (assessed inalbumin)Dye reagent is complex is stable for approximately

one hour


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DisadvantagesNon-linear standard curve over wide rangesResponse to different proteins can vary widely,

choice of standard is very important


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Absorption spectra of anionic and cationic forms of thedye overlap.

So the standard curve is non-linear although all kitproviders of the Bradford assay insist that the assayperforms linearly.

The assay performs linearly over short concentrationstretches.

If your sample is more than 20 micrograms, a secondorder curve will fit much better than a linear curve.


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Points to remember:

Why measure absorbance at 595nm?

When the dye reacts with the protein(protein-dye complex), a

blue-colored solution will be formed which optimally absorbs

light at such wavelength Why should incubation not exceed 1 hour?

The dye interacts with the protein resulting to formation of

precipitates which might give an inaccurate absorbance

reading

What is the relationship of the absorbance reading

with the protein concentration? Directly proportional

What is the identity of the standards? bovine -globulins


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Hydrophobic Interaction Chromatography

Hydrophobic interaction - association of nonpolar

molecules that minimizes exposure to the polar

surroundings

Chromatography - a technique used in which amixture of dissolved components is fractionated as it

moves through a certain type of porous matrix


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HIC

Alternatives

Gel filtration chromatography

Ion exchange chromatography

Reverse phase chromatography

Why HIC?

Different basis of separation

Weaker interactions Less structural damage

Maintain high activity


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Separation principles


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HIC: Purpose

Downstream purification

Separation of biomolecules

Exploits differences in hydrophobicity.

Number of hydrophobic amino acids.

Distribution of these amino acids.


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HIC: Principle

Separation of substances is based on their

varying strength of interaction with

hydrophobic groups attached to an uncharged

gel matrix

Hydrophobic groups on proteins are sufficiently

exposed to bind to the hydrophobic groups on

the matrix. How is this achieved?


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HIC: Principle


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HIC: Principle


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HIC: Applications

HIC has found many applications in the purification of

a wide variety of proteins, including

Monoclonal antibodies Vaccines

Truncated forms of r-proteins

Interferons EGF (epidermal growth factor)

Human growth hormone


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HIC: Advantages

Large volume of sample can be loaded

Samples with high ionic strength can be used

Well suited to use before gel filtration, ion-

exchange and affinity chromatography

Sample eluted with low salt

Purification steps that generate large sample

volume can be coupled with this method


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HIC: Advantages

Good for samples after ammonium sulfate

fractionation.

Sample can be used in ion exchange

chromatography step


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Factors affecting HIC

Type and concentration of ligand

Type of base matrix

Type and concentration of salt

pH

Temperature

Additives


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Buffers

Equilibration buffer, 2 M (NH4)2SO4- used to prime the

column for the binding of proteins

Binding buffer, 4 M (NH4)2SO4- hydrophobic region is

more exposed causing it to interact and bind with thehydrophobic regions of the matrix

Wash buffer, 1.3 M (NH4)2SO4-use - wash the weakly

associated proteins from the column, while the strongly

hydrophobic proteins are bound to the matrixElution buffer, 10 mM Tris/EDTA, -wash the strongly

hydrophobic proteins from the column


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Thus in the fractions obtained

1st fraction: hydrophilic proteins

2nd fraction: the rest of hydrophilic plus some

hydrophobic proteins 3rd fraction: strongly hydrophobic proteins

the last fraction that has the highest absorbance

reading contains the most hydrophobic proteins


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Enzyme-linked Immunosorbent Assay

ELISA


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What is an ELISA?

Enzyme-linked immunosorbent assay

Name suggests three components

Antibody

Allows for specific detection of analyte of interest Solid phase (sorbent) Allows one to wash away all the material that is not

specifically captured

Enzymatic amplification Allows you to turn a little capture into a visible color change

that can be quantified using an absorbance plate reader


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What is it used for?

Measure antibody levels (allergies, vaccines)

Detect viruses (hepatitis, HIV, venereal diseases)

Detect hormonal changes (pregnancy)

Detect circulatory inflammatory markers (cytokines)


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Advantages

Sensitivity

Quantitative

Reproducible

Kit format

Relative sensitivities of tests (approx)

Usual operating range

[Ab] or [Ag]

precipitation

immunoelectrophoresis

double/radial diffusion

10 g/ml - 1 mg/ml

immunofluorescence 0.1 - 10 g/ml

ELISA (colour) 0.1 - 10 ng/ml

(chemiluminescence) 0.01 - 10 ng/ml

radioimmunoassay 0.01 - 10 ng/ml


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Enzymes with Chromogenic Substrates

High molar extinction coefficient (i.e., strong color

change)

Strong binding between enzyme and substrate (low

KM) Linear relationship between color intensity and

[enzyme]


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Antibodies

Specificity Diversityhypervariable region (2020 ~ 1026

combinations; humans make ~ 108)

Affinityrange 105 < K < 109 M-1


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Capture and Detection Antibodies


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ELISA

Enzyme Linked Immunosorbent Assay (ELISA)

Term Was Coined By Engvall and Pearlmann in 1971

Different Types

Sandwich

Indirect

Competitive

Similar To RIA(radio-immuno assay), Except No Radiolabel Can Be Used To Detect Both Antibody and Antigen

Very Sensitive, pg/mL

Relies on Monoclonal Abs


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ELISA-Types


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ELISA

Enzyme-linked immunosorbent assay


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ELISA: In the experiment..

REAGENTS:

Ag: chicken -globulin

1Ab: rabbit anti-chicken polyclonal Ab

2Ab: goat Ab conjugated to horseradish

peroxidase(HRP)

-substrate: 3,3,5,5-tetrametylbenzidine(TMB)


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ELISA: In the experiment


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ELISA-In the experiment..

SPECIFICS:

1. Wash buffer: to discard unbounded Ag

2. Incubation: to allow the Abs react with the Ag

3. HRP enzyme substrate- gives color

*microtiter plates: polystyrene


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ELISA-In the experiment..

RESULTS

amount of colored product is proportional to the

amount of enzyme-linked antibody that binds, which is

directly related to the amount of antibody that waspresent to bind antigen or antigen that was present to

bind antibody

If known amounts of antigen or antibody are added,

a standard curve can be constructed which will allow

the amount of unknown antigen or antibody to be

determined

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Proteins (Isolation Techniques).pdf