CELL AND MOLECULAR BIOLOGY

PROTEINS
Bio150 Lab
Prepared by: Jassy Mary S. Lazarte & Jeremy G. Vicencio Department of Biology, University of the Philippines Manila

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 living cells Our genes code for proteins Proteins are polymers of amino acids

Proteins are

Polypeptides + (cofactors, coenzymes, prosthetic groups, other modifications)

Polypeptides are covalently linked -amino acids Cofactors are non-amino acid components e.g. metal ions like Zn2+ in carboxypeptidase Coenzymes are organic cofactors e.g. nucleotides in lactate dehydrogenase Prosthetic groups are covalently attached cofactors e.g. heme in myoglobin

Proteins and their roles

Enzymes (biological catalysts) Hormones Storage proteins Transport proteins Structural proteins Protective proteins Contractile proteins Toxic proteins

Proteins and their structure

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

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 in vacuole resemble those present in Lysosomes of animal cells other organelles also have proteases

Protein Isolation

How to prevent protein degradation by proteases

the

protein isolation is carried out at low temperature to minimize the activities of these protease to further optimize the results, use the proteases inhibitors

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

Protein Quantitation
A. Colorimetric methods: 1. Biuret 2. Lowry 3. Bradford B. 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)

Protein Quantitation

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 absorb UV light

Protein Quantitation

A260/A280 has high sensitivity for nucleic acid contamination in protein:

% NA A260/A280

A260/A280 lacks sensitivity for protein contamination in nucleic acids:

% protein A260/A280

% protein

% NA

100 95 90 70

0 5 10 30

0.57 1.06 1.32 1.73

100 95 90 70

0 5 10 30

2.00 1.99 1.98 1.94

Protein Quantitation

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 methods

1. Cheap cuvette! (cheap glass or plastic versus quartz) 2. Not contaminating absorbance from nucleic acids!

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 at 465nm) binds to proteins and to absorb strongly at 595nm. The assay is sensitive, but somewhat non-linear

Dye-Binding ( Bradford ) Assay

CBBG primarily responds to
arginine 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 the anionic form Absorbance maximum at 595 nm (blue) The free dye in solution is in the cationic form, Absorbance maximum at 470 nm (red).

Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976. Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).

Dye-Binding ( Bradford ) Assay

Advantages
Fast

and inexpensive Highly 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 in albumin) Dye reagent is complex is stable for approximately one hour

Dye-Binding ( Bradford ) Assay

Disadvantages
Non-linear

standard curve over wide ranges Response to different proteins can vary widely, choice of standard is very important

Dye-Binding ( Bradford ) Assay

Absorption spectra of anionic and cationic forms of the dye overlap. So the standard curve is non-linear although all kit providers of the Bradford assay insist that the assay performs linearly. The assay performs linearly over short concentration stretches. If your sample is more than 20 micrograms, a second order curve will fit much better than a linear curve.

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

Hydrophobic Interaction Chromatography

Hydrophobic interaction - association of nonpolar molecules that minimizes exposure to the polar surroundings Chromatography - a technique used in which a mixture of dissolved components is fractionated as it moves through a certain type of porous matrix

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

Separation principles

HIC: Purpose

Downstream purification Separation of biomolecules Exploits differences in hydrophobicity. Number of hydrophobic amino acids.

Distribution of these amino acids.

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?

HIC: Principle

HIC: Principle

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

HIC: Advantages

Large volume of sample can be loaded Samples with high ionic strength can be used Well suited to use before gel filtration, ionexchange and affinity chromatography Sample eluted with low salt Purification steps that generate large sample volume can be coupled with this method

HIC: Advantages

Good for samples after ammonium sulfate fractionation. Sample can be used in ion exchange chromatography step

Factors affecting HIC

Type

and concentration of ligand Type of base matrix Type and concentration of salt pH Temperature Additives

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 the hydrophobic 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 matrix Elution buffer, 10 mM Tris/EDTA, -wash the strongly hydrophobic proteins from the column

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

ELISA
Enzyme-linked Immunosorbent Assay

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
Allows

amplification

you to turn a little capture into a visible color change that can be quantified using an absorbance plate reader

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)

Advantages

Sensitivity Quantitative Reproducible Kit format

Relative sensitivities of tests (approx)

Usual operating range [Ab] or [Ag] precipitation immunoelectrophoresis double/radial diffusion immunofluorescence ELISA (colour) (chemiluminescence) radioimmunoassay

10 g/ml - 1 mg/ml

0.1 - 10 g/ml 0.1 - 10 ng/ml 0.01 - 10 ng/ml 0.01 - 10 ng/ml

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]

Antibodies

Specificity Diversity hypervariable region (2020 ~ 1026 combinations; humans make ~ 108) Affinity range 105 < K < 109 M-1

Capture and Detection Antibodies

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

ELISA-Types

ELISA

Enzyme-linked immunosorbent assay

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)

ELISA: In the experiment

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

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 was present 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