PROTEIN ANALYSISMETHODS;

WESTERN BLOT and ELISA

Bülent Gögebakan Ph.D.

Gaziantep Üniversitesi Tıp Fakültesi, Göğüs Hastalıkları AD, Hücre Kültürü Laboratuarı

I declare no financial or commercial conflict of interest.

PROTEİN ANALİZ YÖNTEMLERİ

ELISA “Enzyme-linked immunosorbent assay”

Western BlotElectrophoresisChromatography Protein fingerprintX-ray CrystallographyNuclear Magnetic Resonance(NMR)Mass spectrometry(MS)Protein microarray

Protein-Antibody interaction

Proteins 3D3D; antigenic

determinant

ELISA

ELISA (Enzyme-Linked ImmunoSorbent Assay)

A Biochemical-Immunologic methodFor detection of antigen and

antibody amounts in samplesVery sensitive method (pg/mL)

ResearchSerum Antibody Concentration Potential food allergies

◦(Milk, peanut, walnut, almond, egg vb.)Detection of Epidemics

◦HIV, avian flu ,flu , cholera etc. Antigen determination

◦Prenancy hormones, drug allergies, mad cow disease etc.

Determination previously encountered antigens in blood. ◦HIV, avian flu etc.

Where it is used?

What samples suitable to ELISA?

Body Fluids◦Serum◦Plazma◦BAL◦Plevra◦Breath air etc.

Cell culture◦Supernatant ◦Cell lysate

Tissue (homogenization..!)

Types of ELISA

Competitive ELISA

Sandwich ELISA

Cover plates with spesific antibodies

Sandwich ELISA

Incubation with your sample that contains protein of interest

Incubation with enzyme-linked secondary antibody

Sandwich ELISA

Addition of substrate and color change

Sandwich ELISA

Indirect ELISA

Study schedule ◦Samples

Serum Blood plasma Cell line Breath air etc.

Dilution/CondensationSelection of ELISA Kit

Sensitivity

Selection of suitable tools ◦Pipette◦Tips

Carefull about?

Western Blot

Western Blot (WB) is used in molecular biology to detect the presence of individual proteins and amount of proteins in any protein sample

Southern Blot; (1975) (DNA/DNA-probe)Western Blot; Towbin (1979)

WB Nerelerde Kullanılır?

Cancer biology ve patology MicrobiologyImmunologyProtein BiochemistryTissue studiesAntibody Assays

WB

WB

Protein Isolation and SDS-PAGE

•Protein İzolasyonu• Homojenizasyon (doku)• Proteaz İnhibitörleri• Liziz bufer

•SDS-PAGE• Her bir kuyucuğa aynı

miktar protein• Jel konsantrasyonu

(%Akrilamit-bisakrilamit)• Elektroforez süresi ve akımı

SDS-PAGE and Blotting

Blotting

VISUALISATION

Carefull about?

Protein ◦ Isolation (Cell culture/Tissue)◦ Determination of amaount

SDS-PAGE ◦ Gel concentration◦ Electrophoresis duration and current

Blotting Selection of antibody

◦ Protein of interest can be recognised with several antibodies

Optimization Time of incubation with primary antibody and

temperature

ELISA - Western Blot

In each method;◦Spesific proteins can be determined

from tissue or cell lysates◦Antibodies used (primary ve secondary)◦Must be used for determination of

novel proteins and their functions

Alternative Methods

Flow cytometry

Luminex

Flow cytometry

Flow cytometry

Flow cytometry

Flow cytometry

Luminex

Luminex

Luminex 200, Luminex Corp.

635 nm

532 nm Bead dye

RPE

Thank you...