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

At _______, amino acid residues with aromatic rings in a protein absorb light

Beer-Lambert law

UV SPECTROSCOPY METHOD follows the ____________________ in which absorbance is proportional to concentration and path length

tryptophan
tyrosine

phenylalanine

The amino acids ___________ and ___________ contribute most to the absorbance at this wavelength, while ____________ (λmax 279 nm) makes a minor contribution

20-3000μg

UV SPECTROSCOPY METHOD can be used to detect protein in the _____________ range

Peptide bond

Determination of protein concentration by measurement of absorbance at 205nm (A205) is based on absorbance by the?

1-100μg/mL

UV Spectroscopy Measurement can be used to quantitate protein solutions with concentrations of _____________ protein

Greater DAN :))))

The absorptivity for a given protein at 205nm is several-fold (GREATER THAN or LESS THAN) that at 280 nm

Protein concentration

Can be determined by measuring the intrinsic fluorescence based on fluorescence emission by the aromatic amino acids tryptophan, tyrosine, and phenylalanine

205 nm

Absorbance at ________ can be used to quantitate dilute solutions, or for short path length applications

5-50μg/mL

Spectrofluorometric Measurement can be used to quantitate protein solutions with concentrations of?

280 nm

320 and 350 nm

Spectrofluorometric Measurement:

Normally, the excitation wavelength is set to _________ and the emission wavelength is between _____ and ______ nm

Protein

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

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

Commercially available standards in COLORIMETRIC PROTEIN ASSAY TECHNIQUES

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

COLORIMETRIC PROTEIN ASSAY TECHNIQUES

Biuret reaction

This colorimetric technique is based on the complex formation of cupric ions with proteins

Lowry Method

This colorimetric technique is based on the biuret reaction with additional steps and reagents to increase the sensitivity of detection

purplish-violet

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

copper sulfate

In this Biuret reaction, ______________ is added to a protein solution in strong alkaline solution = (+) purplish-violet color

FolinCiocalteu reagent

Lowry adds phosphomolybdic/phosphotungstic acid also known as _________________

blue-green color

FolinCiocalteu reagent = (+) _____________ (color) @ 650nm-750nm

Coomassie brilliant blue G-250

Bradford Method uses the binding of _______________________ dye to proteins

465 nm

acidic

The Coomassie brilliant blue G-250 dye is protonated and is reddish/brown with an absorbance maximum of __________ at _________ pH

0.2-20 μg

In Bradford Method, _____________ of protein can be detected

deep blue

562nm

The bicinchoninic acid cuprous complex = (+) _______ color at _________ (absorbance)

1. Microplate Detection Method
2. Cuvette Detection Method

FLUORESCENT DYE-BASED ASSAYS

0.05-25 μg

The range of Microplate Detection Method is ___________

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:

3-(4 carboxybenzoyl)quinoline-2-carboxyaldehyde (CBQCA)

require the addition of a thiol (2-mercaptoethanol) or cyanide

2- mercaptoethanol or 3-mercaptopropionic acid

OPA reacts with primary amino acid (except cysteine) in the presence of ______________________ to form a highly fluorescent adduct

proline and 4-hydroxyproline

OPA also reacts with 4-amino-1-butanol and 4-aminobutane-1,3-diol produced from oxidation of ________ and ____________

340 nm and 455 nm

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

COLUMN CHROMATOGRAPHY

this method uses a glass or plastic tube 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

column matrix

also known as resin consists of microscopic beads of inert material

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

2

Each sodium dodecyl sulfate contributes to how many negative charges?

SDS

a detergent that disrupts protein tertiary structure

negative

SDS coats all the polypeptides with (positive or negative) charges

a. It coats all the polypeptides with negative charges
b. It masks the natural charges of the subunits

SDS has two advantages:

RIGHT <3333

RIGHT OR WRONG???


The electrophoretic mobility of proteins upon SDS-PAGE is inversely proportional to the logarithm of the protein's molecular weight

SDS-PAGE

often used to determine the molecular weight of proteins

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

Three high-sensitivity colorimetric staining methods can be used either directly after electrophoresis:

Coomassie blue staining

This is an anionic triphenylmethane dye

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

Fluorescence Staining Method

In Gel Staining After Electrophoresis, this is considered as endpoint method thus the results are highly reproducible

fluorescence imager

Fluorescence Staining Method require a __________ for visualization and that they are significantly more expensive than classical colorimetric dyes

proteomics

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

2DE gel coupled with MS

most reproducible and effective technology to separate overall proteins of microorganisms, cells, and heterogeneous tissues

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

2 Gel-based proteomics

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

2 Gel-free proteomics

1. Net charge at different pH
2. Molecular weights determined by SDS- PAGE

In 2DE, the complex protein samples are separated in two dimensions according to their:

1. Conventional Isoelectric Focusing
2. Immobilized pH Gradient (IPG)
3. Nonequilibrium pH Gel Electrophoresis

First Dimension Electrophoresis (3)

Conventional Isoelectric Focusing

the complex protein samples are separated according to their net charge at different pH

ampholytes

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

Isoelectric point (pI)

The pH at which the protein has no net charge

Immobilized pH Gradient (IPG)

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

Nonequilibrium pH Gel Electrophoresis

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

Laemmli buffer or Tris-Tricine buffer

The second step of 2DE separates proteins based on their molecular weight using a vertical electrophoretic device with either _________ or _________ (buffers)

CyDye
DIGE
Fluors

charge-matched cyanine dyes

Cy2
Cy3
Cy5

multiplexed fluorescent dyes

2D-DIGE

can run more than one sample (maximum 3 samples) on a single gel at once to address the issue of gel-to-gel variability

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

SAMPLE BUFFER CONSTITUENTS FOR 2DE

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 _________

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

CAPILLARY ELECTROPHORESIS

Typical gel-free technique, with the advantage of superior separation efficiency, small sample consumption, short analysis time, and automatability

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)

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

Protein separation in Capillary Electrophoresis is based on a variety of properties:

Microchip Electrophoresis

Miniaturized form of conventional Capillary Electrophoresis

5 cm to 15 cm

Microchip Electrophoresis could integrate injection, separation, and detection on a single microchip with typical channel lengths of ____ cm to ____ cm

Microchip Electrophoresis

enables fast separation of protein, peptide, and single-cell analysis, which are normally present in small amounts in complex matrices

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

Capillary Electrochromatography

A hybrid technique of HPLC and CE
It combines the high peak efficiency that is characteristic of electrically driven separations with high separation selectivity

MASS SPECTROMETER

Exploit the difference in the mass-to-charge (m/z) ratio of ionized atoms or molecules to separate them from each other

Electrospray ionization MS (ESI-MS)

Matrix-assisted laser desorption/ionization-time of flight MS (MALDI-TOF MS)

The two most prominent MS methods for protein analysis are?

protein

The immobilized target for a western blot is?

Dithiothreitol or 2-mercaptoethanol

may also be used to separate proteins into subunits

denaturant

Before loading, the sample is treated with __________

WESTERN BLOT

Uses pre-stained molecular-weight standards such as cytochrome C (11,700 d) to myosin (205,000 d)

Nitrocellulose

has a high affinity for proteins and is easily treated with detergent with 5% dry milk to prevent binding of the primary antibody probe to the membrane itself (blocking) before hybridization

PVDF and anion (DEAE)

Cation (CM) exchange cellulose

Other membrane types used for protein blotting are? (2)

Western blot protein probes

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

Polyclonal antibodies

are comprised of a mixture of immunoglobulins directed at more than one epitope on the antigen

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

1. 35S
2. Horseradish peroxidase (HRP) or alkaline phosphatase (AP)

The protein probes used in western blot applications may be labeled with:

ELISA

Application of the western blot method is confirmation of___________________ results for human immunodeficiency virus (HIV) and hepatitis C virus (HCV)