Post on 14-Jan-2016
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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...