MODX FINALS TECHNIQUES IN PROTEIN QUANTIFICATION & ANALYSIS

Protein with Aromatic rings absorb light at

280nm

Proteins with aromatic rings
T,T,P

phenylalanine, tryptophan, and tyrosine

more universally applicable assay than A280

Detecting Proteins With Absorbance at 205nm (A205)

assay based on absorbance by the peptide bond

Detecting Proteins With Absorbance at 205nm (A205)

A205 quantitate protein with concentrations of

100μg/mL protein

A280 quantitate protein with concentrations of

20–3000μg range

used to quantitate protein solutions with concentrations of 5–50μg/mL

Detecting Proteins by Intrinsic Fluorescence Emission

Commercially available standards

BSA
BGG
igG

a. Bovine serum albumin (BSA)*

b. Bovine gamma globulins

c. Immunoglobulins

Postive result of Biuret reaction

purplish-violet color

most or well known colorimetric assay technique

Biuret reaction

Positive result of Lowry Method

(+) blue-green color @ 650nm-750nm

The protein detection range of Lowry Method

5–100μg

Reagent added in lowry method

Folin-Ciocalteu reagent

dye that reacts primarily with arginine and to a lesser extent with lysine, histidine, tyrosine, tryptophan, and phenylalanine residues when under acidic conditions

Coomassie brilliant blue G-250 dye

Positive result of Bradford Method

blue color with an absorbance maximum at 595nm

easiest and fastest of the protein determination method

Bradford Method

_________ reacts with the cuprous ion generated by the biuret reaction under alkaline conditions.

sodium salt of bicinchoninic acid

Positive result of Bicinchoninic Acid Assay (BCA) Method

(+) deep blue color at 562nm

modified biuret reaction

Bicinchoninic Acid Assay (BCA) Method

The protein detection range of Bradford Method

0.2–20 μg of protein can be detected

The protein detection range of Bicinchoninic Acid Assay (BCA) Method

Detection range is 0.2–50μg

This technique can be used to

quantitate proteins and peptides

containing either lysine or a free N

terminus

Microplate Detection Method

The protein detection range of Microplate Detection Method

0.05–25 μg

Three dyes used to quantitate proteins, or

amino acids in a microplate format

O,F,3

Ophthalaldehyde (OPA)
Fluorescamine
3-(4carboxybenzoyl)quinoline-2-

carboxyaldehyde (CBQCA)

Fluorescence of OPA derivatives is monitored at

emission wavelengths of 340 nm and 455 nm

most common method for purifying

proteins from other protein molecules

COLUMN CHROMATOGRAPHY

This chromatography can separate proteins

based on the size and shape

Size Exclusion Chromatography

separate proteins according to their surface charges

Ion-Exchange Chromatography

protein binds to a molecule for which it has specific affinity

Affinity Chromatography

Three high-sensitivity colorimetric staining

methods can be used either directly after

electrophoresis

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

also called as negative staining

Zinc-reverse staining

Zinc-reverse staining uses _____ for protein detection in electrophoresis gels

imidazole and zinc salts

Considered as endpoint method

Fluorescence Staining Method

most commonly used dye in Fluorescence Staining Method

CyDyes

In 2DE, the complex protein samples are

separated in two dimensions according to their

Net charge at different pH
Molecular weights determined by SDSPAGE

narrow tube gel containing molecules called

ampholytes

Most popular mode of chromatography

Reversed-Phase High Performance Liquid Chromatography

Chromatographic method capable

of separating biochemical mixtures

of highly specific nature

Affinity High Performance Liquid Chromatography

method of choice for separation of biomolecules

Gel-Permeation HPLC

Advanced version of reversedphase-

HPLC

Ligand-Exchange HPLC

A hybrid technique of HPLC and CE

Capillary Electrochromatography