(MODX FINALS) TECHNIQUES IN PROTEIN QUANTIFICATION & ANALYSIS

1. UV absorbance methods
2. Dye-binding assays using colorimetric- based detection
3. Dye-binding assays using fluorescent- based detection

Three major types of spectrophotometric assays:

280nm

UV SPECTROSCOPY METHOD:

Detecting Proteins With Absorbance at ___________

Beer-Lambert law

The method follows the ____________ in which absorbance is proportional to concentration and path length

tryptophan
tyrosine

The amino acids ______ (λmax 279.8nm) and _______ (λmax 274.6 nm) contribute most to the absorbance at this wavelength, while phenylalanine (λmax 279 nm) makes a minor contribution

205nm (A205)

is based on absorbance by the peptide bond

tryptophan
tyrosine
phenylalanine

the aromatic amino acids

280 nm
320 and 350 nm

Spectrofluorometric Measurement:

Normally, the excitation wavelength is set to ______nm and the emission wavelength is between ______nm and _____nm

COLORIMETRIC PROTEIN ASSAY TECHNIQUES

All colorimetric protein assays require protein standard to estimate the concentration of a sample

1. Bovine serum albumin (BSA)
2. Bovine gamma globulins
3. Immunoglobulins

Commercially available standards :

1. Biuret reaction
2. Lowry Method
3. Bradford Method
4. Bicinchoninic Acid Assay (BCA) Method

COLORIMETRIC PROTEIN ASSAY TECHNIQUES

Biuret reaction

based on the complex formation of cupric ions with proteins.

purplish-violet color

Biuret reaction

In this reaction, copper sulfate is added to a protein solution in strong alkaline solution = (+) _____ color

Lowry Method

based on the biuret reaction with additional steps and reagents to increase the sensitivity of detection

blue-green color @ 650nm-750nm

Lowry adds phosphomolybdic/phosphotungstic acid also known as Folin Ciocalteu reagent = (+) _____color @ _____nm-____nm

5-100μg

Lowry Method
The protein detection range is

Coomassie brilliant blue

Bradford Method

uses the binding of ____________ G-250 dye to proteins

465 nm at acidic pH

reddish/brown with an absorbance maximum of

0.2-20 μg

Bradford Method
_________ of protein can be detected

cuprous ion

based on the fact that the sodium salt of bicinchoninic acid reacts with the __________ generated by the biuret reaction under alkaline conditions.

deep blue color st 562nm

The bicinchoninic acid cuprous complex (+)

0.2-50μg

Bicinchoninic Acid Assay (BCA) Method

Detection range is

1. Microplate Detection Method
2. Cuvette Detection Method

FLUORESCENT DYE-BASED ASSAYS

Microplate Detection Method

This technique can be used to quantitate proteins and peptides containing either lysine or a free N terminus

1. Ophthalaldehyde (OPA)*
2. Fluorescamine
3. 3-(4 carboxybenzoyl)quinoline-2 carboxyaldehyde (CBQCA)*

Three dyes used to quantitate proteins, or amino acids in a microplate format:

Cuvette Detection Method

OPA reacts with primary amino acid (except cysteine) in the presence of 2 mercaptoethanol or 3-mercaptopropionic acid to form a highly fluorescent adduct

340 nm and 455 nm

Cuvette Detection Method

Fluorescence of OPA derivatives is monitored at excitation and emission wavelengths of ____nm and ___nm

COLUMN CHROMATOGRAPHY

The most common method for purifying proteins from other protein molecules

glass or plastic tube

COLUMN CHROMATOGRAPHY

this method uses a ___________ filled with resin that can separate proteins based on their physical properties as they flow through the column

Size Exclusion Chromatography

This chromatography can separate proteins based on the size and shape on a gel filtration column

Ion-Exchange Chromatography

Uses a resin to separate proteins according to their surface charges

Ion-Exchange Chromatography

This type of column contains a resin bearing either positively or negatively charged chemical groups

Affinity Chromatography

Uses the principle that the protein binds to a molecule for which it has specific affinity

Affinity Chromatography

molecule can be immobilized through covalent attachment to the resin in a column

Affinity Chromatography

The power of affinity chromatography lies in the specificity of binding between the affinity reagent on the resin and the molecule to be purified

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The hydrophobic tail of SDS interacts strongly with polypeptide chains

SDS-PAGE

is often used to determine the molecular weight of proteins

1. Coomassie blue staining
2. Zinc-reverse staining
3. Silver staining

Gel Staining After Electrophoresis: Colorimetric Staining Methods - 3

Coomassie R-250 (Red tint)
Coomassie G-250 (Green tint)

anionic triphenylmethane dye Modifications: red and green

Zinc-reverse staining

also called as negative staining which uses imidazole and zinc salts for protein detection in electrophoresis gels

Silver staining

based on the binding of silver ions to the proteins followed by reduction to free silver, sensitization, and enhancement

proteomics

The field that uses techniques to resolve thousands of polypeptides is called

1. Two-dimensional gel electrophoresis (2DE) gel
2. 2DE fluorescence differential imaging gel electrophoresis (DIGE)

Gel-based proteomics: - 2

1. Liquid chromatography (LC)
2. Capillary electrophoresis (CE)

Gel-free proteomics - 2

ampholytes

the mixture of proteins is electrophoresed through a narrow tube gel containing molecules called

Isoelectric point (pI)

The pH at which the protein has no net charge

Isoelectric point (pI)

Protein is no longer drawn toward the anode or the cathode à STOPS

Immobilized pH Gradient (IPG)

IPG is an integrated part of a polyacrylamide gel matrix fixed on a plastic strip

Nonequilibrium pH Gel Electrophoresis

_____ technique can resolve proteins with basic to extremely high pI (7.0-11.0) that cannot be separated by traditional method

1. Chaotropic Agents (8-9M Urea)
2. Detergents
3. Reducing Agents (DTT)
4. Carrier Ampholytes

SAMPLE BUFFER CONSTITUENTS FOR 2DE - 4

Chaotropic Agents (8-9M Urea)

It effectively disrupts noncovalent and ionic bonds between amino acid residues

Detergents

Hydrophobic interactions within a polypeptide chain or between proteins in protein complexes can be disrupted in the presence of detergents and it can also increase solubility, especially of membrane proteins

Reducing Agents (DTT)

cleave disulfide bond cross-links within and between protein subunits, thereby promoting protein unfolding and maintaining proteins in their fully reduced states

Carrier Ampholytes

They can reduce protein-matrix hydrophobic interactions and usually included at a concentration of 0.5%-2% (v/v) in sample solutions for IPG strip focusing

a. Molecular mass (SDS-capillary gel electrophoresis [CGE])
b. Isoelectric point (capillary isoelectric focusing [CIEF])
c. Electrophoretic mobility (capillary zone electrophoresis [CZE])

GEL FREE PROTEOMICS TECHNIQUES: CAPILLARY ELECTROPHORESIS - 3

Molecular mass (SDS-capillary gel electrophoresis [CGE])

a capillary is filled with gel or viscous solution (to form molecular sieve) and the electroosmotic flow (EOF) is usually suppressed

Electrophoretic mobility (capillary zone electrophoresis [CZE])

sample is introduced into a buffer-filled capillary either electrokinetically (with low voltage) or hydrodynamically (with pressure or suction)

LIQUID CHROMATOGRAPHY

Useful in proteomics and genome research because it can detect molecules at the nanomolar level

a. Reversed-phase High performance liquid chromatography (HPLC)
b. Affinity HPLC
c. Gel permeation HPLC
d. Ligand-exchange HPLC
e. Capillary HPLC

Types of Liquid chromatography

Reversed-Phase HPLC

Most popular mode of chromatography because of its wide range of applications and the availability of various mobile and stationary phases

Affinity HPLC

Chromatographic method capable of separating biochemical mixtures of highly specific nature

Gel-Permeation HPLC

Works on the principle of sizes of the compounds, which is the same as the size exclusion chromatography

Gel-Permeation HPLC

It is a method of choice for separation of biomolecules such as peptides, proteins, and enzymes

Ligand-Exchange HPLC

Advanced version of reversed-phase-HPLC where the reversed-phase column is replaced by an ion-exchange column

Ligand-Exchange HPLC

It has been used widely for the analysis of all inorganic and organic ionic species

Capillary Electrochromatography

A hybrid technique of HPLC and CE

Capillary Electrochromatography

It combines the high peak efficiency that is characteristic of electrically driven separations with high separation selectivity

Western blot protein probes

are antibodies (polyclonal or monoclonal) that bind specifically to the immobilized target protein

Polyclonal antibodies

can give a more robust signal, especially if the target epitopes are partially lost during electrophoresis and transfer

Monoclonal antibodies

are more specific and may give less background noise

ELISA CONFIRMATION

pplication of the western blot method is confirmation of enzyme-linked immunoassay (ELISA) results for human immunodeficiency virus (HIV) and hepatitis C virus (HCV)

ELISA

Screening test for HIV and HCV

Western Blot

Confirmatory test for HIV and HCV

• 35s

• Horseradish peroxidase (HRP) or

• Alkaline phosphatase (AP)

The protein probes used in western

Detection signal

blot applications may be labeled with: