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PROTEIN ANALYSIS METHODS; WESTERN BLOT and ELISA

PROTEIN ANALYSIS METHODS; 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.

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PROTEIN ANALYSIS METHODS; WESTERN BLOT and ELISA

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  1. PROTEIN ANALYSISMETHODS; WESTERN BLOT and ELISA Bülent GögebakanPh.D. Gaziantep Üniversitesi Tıp Fakültesi, Göğüs Hastalıkları AD, Hücre Kültürü Laboratuarı

  2. Ideclareno financial or commercial conflict ofinterest.

  3. PROTEİN ANALİZ YÖNTEMLERİ • ELISA “Enzyme-linkedimmunosorbentassay” • Western Blot • Electrophoresis • Chromatography • Protein fingerprint • X-ray Crystallography • NuclearMagneticResonance(NMR) • Massspectrometry(MS) • Protein microarray

  4. Protein-Antibodyinteraction • Proteins 3D • 3D; antigenic determinant

  5. ELISA • ELISA (Enzyme-LinkedImmunoSorbentAssay) • A Biochemical-Immunologicmethod • Fordetection of antigenandantibodyamounts in samples • Verysensitivemethod (pg/mL)

  6. Where it is used? • Research • Serum AntibodyConcentration • Potentialfoodallergies • (Milk, peanut, walnut, almond, eggvb.) • Detection of Epidemics • HIV, avianflu ,flu, choleraetc. • Antigendetermination • Prenancyhormones, drugallergies, mad cowdiseaseetc. • Determinationpreviouslyencounteredantigens in blood. • HIV, avianfluetc.

  7. Whatsamplessuitableto ELISA? • Body Fluids • Serum • Plazma • BAL • Plevra • Breathair etc. • Cell culture • Supernatant • Cell lysate • Tissue (homogenization..!)

  8. Types of ELISA

  9. Competitive ELISA

  10. Sandwich ELISA Coverplateswithspesificantibodies

  11. Sandwich ELISA • Incubationwithyoursamplethatcontains protein of interest

  12. SandwichELISA • Incubationwithenzyme-linkedsecondaryantibody

  13. SandwichELISA • Addition of substrateandcolorchange

  14. IndirectELISA

  15. Carefullabout? • Studyschedule • Samples • Serum • Bloodplasma • Cellline • Breathairetc. • Dilution/Condensation • Selection of ELISA Kit • Sensitivity • Selection of suitabletools • Pipette • Tips

  16. Western Blot • Western Blot (WB) is usedin molecularbiologytodetectthe presence of individualproteinsandamount of proteins in any protein sample • SouthernBlot; (1975) (DNA/DNA-probe) • Western Blot; Towbin (1979)

  17. WB Nerelerde Kullanılır? • Cancerbiology ve patology • Microbiology • Immunology • Protein Biochemistry • Tissuestudies • AntibodyAssays

  18. WB

  19. WB

  20. Protein Isolationand SDS-PAGE • Protein İzolasyonu • Homojenizasyon (doku) • Proteaz İnhibitörleri • Lizizbufer • SDS-PAGE • Her bir kuyucuğa aynı miktar protein • Jel konsantrasyonu (%Akrilamit-bisakrilamit) • Elektroforez süresi ve akımı

  21. SDS-PAGE andBlotting

  22. Blotting

  23. VISUALISATION

  24. Carefullabout? • Protein • Isolation (Cell culture/Tissue) • Determination of amaount • SDS-PAGE • Gel concentration • Electrophoresisdurationandcurrent • Blotting • Selection of antibody • Protein of interest can be recognisedwithseveralantibodies • Optimization • Time of incubationwithprimaryantibodyandtemperature

  25. ELISA - Western Blot • Ineachmethod; • Spesificproteins can be determinedfromtissueorcelllysates • Antibodiesused(primary ve secondary) • Must be usedfordetermination of novelproteinsandtheirfunctions

  26. AlternativeMethods • Flow cytometry • Luminex

  27. Flow cytometry

  28. Flow cytometry

  29. Flow cytometry

  30. Flow cytometry

  31. Luminex

  32. 635 nm RPE 532 nm Bead dye Luminex 200, Luminex Corp. Luminex

  33. Thankyou...

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