Techniques in Protein Quantification and Analysis

blue-green color

In Lowry method, what is the end color when adds phosphomolybdic/phosphotungstic acid

650nm-750nm

In Lowry method, blue-green color reach at what nm?

Proteins

Organic compounds made of amino acids.

Polypeptides

Another name for proteins, consisting of amino acids.

UV Spectroscopy

Method using UV light to measure protein absorbance.

Dye-binding Assays

Techniques using dyes to detect protein concentration.

Intrinsic Fluorescence

Protein concentration can also be determined by measuring the _______ based on fluorescence emitted by aromatic amino acids.

tryptophan, tyrosine, phenylalanine

Amino acids with aromatic rings:

Beer-Lambert Law

Absorbance proportional to concentration and path length.

Colorimetric Assays

Methods detecting protein via color change.

purplish-violet color

In Biuret reaction, what is the end color when copper sulfate is added to a protein solution in strong alkaline solution

Biuret reaction

Based on the complex formation of cuprous ions with proteins.

Lowry Method

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

Bradford Method

Uses Coomassie brilliant blue G-250 dye for protein quantification

reddish/brown

End product in Bradford method

465 nm at acidic pH

Absorbance used what acid environment needed in Bradford method to achieve the reddish/brown color

Ion-Exchange Chromatography

Separates proteins based on surface charge.

deep blue color
562 nm

The bicinchoninic acid cuprous complex end product? And what is the absorbance needed?

Bicinchoninic Acid Assay

Based on the fact that the sodium salt of bicinchoninic acid reacts with the cuprous ion generated by the biuret reaction under alkaline conditions.

Microplate Detection Method

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

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

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

Absorbance at 280nm

Common wavelength for protein quantification.

279.8 nm

Wavelength of tryptophan

274.6 nm

Wavelength of tyrosine

279 nm

Wavelength of phenylalanine

Absorbance at 205nm

Measurement used for detecting proteins with peptide bonds.

A280/A260 Ratio

Used to assess nucleic acid contamination in protein samples.

Fluorescent Adduct

Highly fluorescent compound formed by OPA reaction.

Cation Exchange Media

Negatively charged resin attracting positively charged molecules.

Anion Exchange Media

Positively charged resin attracting negatively charged molecules.

50fmol

Detection limits of amino acids in Cuvette detection method

Column Chromatography

Most common method for purifying proteins based on properties.

Size Exclusion Chromatography

Separates proteins based on size and shape.

resin

The column matrix also known as _____ consists of microscopic beads of inert material

Affinity Chromatography

Purification based on specific protein-ligand binding.

Coomassie blue staining
Zinc-reverse staining
Silver staining

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

Zinc-Reverse Staining

Negative staining using zinc salts for protein detection and acts sometimes as a secondary stain when the proteins are not properly stained by the Coomassie blue stain

Coomassie blue staining

anionic triphenylmethane dye

Silver Staining

Detects proteins via binding and reduction of silver ions.

SDS-PAGE

The hydrophobic tail of SDS interacts strongly with polypeptide chains

SDS

a detergent that disrupts protein tertiary structure

Fluorescence Imager

Device for visualizing fluorescently labeled proteins.

Proteomics

Field studying thousands of polypeptides in samples.

Two-Dimensional Gel Electrophoresis (2DE)

Separates proteins by charge and molecular weight.

Isoelectric Point (pI)

pH where protein has no net charge.

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 called

Reducing Agents

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

Mercaptoethanol
Dithiothreito

What are the two reducing agents:

Carrier Ampholytes

Set up pH gradient in isoelectric focusing.

Gel-Based Proteomics

Techniques using gels to separate proteins.

Gel-Free Proteomics

Methods like liquid chromatography for protein analysis.

inversely proportional

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

Capillary Electrophoresis

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

Immobilized pH Gradient (IPG)

Polyacrylamide gel matrix fixed on plastic strip.

Non-equilibrium pH Gel Electrophoresis (NEPHGE)

Resolves proteins with pI 7.0-11.0 efficiently.

Second Dimension SDS-PAGE

Separates proteins based on molecular weight.

Microchip Electrophoresis

Miniaturized CE integrating injection, separation, detection.

Two-Dimensional Difference Gel Electrophoresis (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

Liquid Chromatography

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

Chaotropic Agents (8-9M Urea)

Disrupt noncovalent bonds in proteins

Detergents

Hydrophobic interactions within a polypeptide chain or between proteins in protein complexes

Triton X 100
Nonidet P40
Sodium Dodecyl Sulfate

Detergents used of 2DE

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

Separates biochemical mixtures using specific interactions.

Gel-Permeation HPLC

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

Ligand-Exchange HPLC

Advanced HPLC using ion-exchange columns.

Capillary Electrochromatography

Combines HPLC and CE for efficient separations.

Western Blot Probes

Antibodies binding specifically to target proteins.

Polyclonal Antibodies

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

Monoclonal Antibodies

Specific antibodies targeting a single epitope.

Mass Spectrometer

Measures mass-to-charge ratio of ionized molecules.

Western Blot

Modification of Southern Blot

Protein

The immobilized target for a western blot is?

35S
Horseradish peroxidase (HRP) or alkaline phosphatase (AP)

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