Protein analysis techniques

Higher Isoelectric point

Have more basic amino acids

Lowr PI values

more acidic amino acids

Isoelectric equation

pI = ^{pK_{COOH +} pK} NH3 / 2

Protein electrophoresis

Separate proteins based on their size and charge

SDS-PAGE

Denatures proteins and coats them with a negative charge

They can be sepaarted by molecular weight and not their shape and charge

SODIUM DODECYL SULFATE ( SDS) :- a reducing agent

Polyacrylamide gel electrophoresis :- separte

NATIVE PAGE

Proteins are separated based on their native confirmation and charge

Reducing agents in SDS- PAGEe

Beta-mercaptoethanol & DIthiothreitol ( DDT)

break sulfide bonds within and protein molecules

Coomassie Brilliant Blue or Silver Stain

To visualize separated proteins

Electrophoresis buffer

To maintain pH and conductivity

Smaller proteins move faster while larger proteins move slower

Proteins migrate toward the anode ( positive electrode)

Western Blotting

Protein separation

Primary antibody :- specific to target protein

Secondary antibody :- conjugated to an enzyme that binds to primary antibody

ELIZA

Uses antibodies to detect and quantify proteins directly in a liquid sample

Southern Blotting

To detect specific DNA sequences

they are separated by size using agarose gel electrophoresis

Northern Blotting

to detect RNA sequences

S DNA

N RNA

O O

W PROTEIN

Chromatography

Analytical technique commonly used for separating a mixture of chemical substances into its individual components

Eluent

fluid entering the column

Eluate

fluid exiting the column

Mobile phase

Higher absorption - slower the molecules will move in stationary phase

Higher solubility in mobile phase ;- faster the molecule will move through the column.

Why do different compounds posses different affinities toward the stationary and mobile phases?

Different compounds have different affinities toward stationary and mobile phases due to variations in their polarity, charge, size, and solubility, affecting how they interact with each phases.

Paper chromatography

separation = polarity of molecules

compound spotted directly on a cellulose paper

Thin Layer chromatography

Base of separation = polarity of molecules

glass is coated with thin layer of silica on which is spotted the compound

Liquid column chromatography

Polarity of molecules

size exclusion chromatography

size of molecules

larger molecules come out first

there is no interaction , physical, chemical between the analyte and stationary phase

Ion- exchange chromatography

ionic charge of the molecles

affinity chromatography

binding affinity of the analyte molecule to the molecule immobilized on stationary phase

Gas chromatography

Boiling point of molecules ( argon or helium)

molecule with the highest boiling point comes out of the column last.

low boiling point comes out of the column first

TLC

component that travels the least distance in TLC is most polar since it binds to the silica most tightly

component that travles the maximum distance is least polar

Retention factor

distance travelled by the individual component divided by total distance travelled by solvent

Lower Rf , more polar the component

distance travelled by compoet = 2

distance travelled by solvent = 5 Rf = 2/5 = 0.4

Ultracentrifugation

separates proteins based on their size, shape, and density using high speed cenriguation

larger protiens will have greater centrifugal force.

sedimentation coefficient

measure of how fast a particle sediments under the influence of centrifugal force.

svedberg units (S) = larger proteins generally have higher sedimentation coeefinets, they sediment faster

Mass spectrometry

identidies and quantifies proteins based on their mass to charge ration ( m/z)

Circular dichroism spectroscpoy

measures absorption of circulary polarized light to analzyse protein secondary strcutures

NMR spectrospiy

structure and dynamics of proteins in soultion

Fourier- Transform Infrared spectroscopy

analyze protein secondary strctures based on infrared absorption spectra.

Two dimensional gel electrophoresis

combines isoelectric foscuing and SDS-PAGE for high resolution protein separation

Surface Plasmon Resonance

measures the interaction between molecles in real time without need for labelling

isotope- coded affinity tags

involves labeling proteins with isotopic tags, which allows for quantification and identification of proteins via mass spectromtery