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  • 1. Technical training CST Michiel van der Sanden
  • 2. Signaaltransductie Effector1 Kinase Phosphospecific antibody PhosphoryleringEffector 3 P Effector 3 Inactief Actief y ibod nt Dephosphorylering la ta To Phosphatase EFFECT
  • 3. CST Produkten• Kinases and Kinase Assays• SiRNA kits• Antibodies
  • 4. SiRNA kit X Kinase Phosphospecific antibody PhosphoryleringSubstrate P Substraat Inactief actief y ibod ant t al To
  • 5. Toepassingen phosphospecifieke en totaal antibodies• Western Blot (WB)• Immunohistochemistry (IHC)• Immunofluorescentie (IF)• Flow cytometry (F)• Immunoprecipitatie (IP)• In Cell Western (ICW)• ELISA
  • 6. Western blot Classical HRP WB LICOR Fluorescent WB - + SP600125 JNK inhibitorp-ATF Actin
  • 7. Western blot protocol• Cell/Tissue sample preparation in buffer• SDS PAGE Gel• Transfer of proteins from Gel to Blot• Block blot with Blocking buffer for 1 h• Incubate blot overnight in BSA/Milk TBS-T• Wash blot 3 X• Incubate secondary antibody 1 h• Do ECL and exposure to blot or use LICOR system
  • 8. Immunohistochemistry/ ImmunofluorescenceImmunohistochemistry Immunofluorescence
  • 9. Immunohistochemistry/ ImmunofluorescenceImmunohistochemistry Immunofluorescence
  • 10. IHC/IF blot protocol• Fixation of samples• Deparaffinization of slides (paraffin sections)• Antigen unmasking (paraffin section)• Washing of samples• Blocking of samples• Incubation primary antibody overnight• Washing of samples• Incubation secondary antibody 1 hour• Washing• ABC/DAB staining (IHC)• Mount slides (fixing them on object glas)• Analyze them under microscope
  • 11. Flow cytometryGrote Voordeel Flow: Single cell measurement + quantificatie
  • 12. Flow blot protocol• Collection of samples• Fixation of cells• Permeabilization of cells• Aliquot and incubate cells in incubation buffer• Incubation primary antibody 30-60 minutes• Washing of samples• Incubation secondary antibody 30 minutes (if necessary)• Washing• Analyze cells on flow cytometric unit
  • 13. In Cell Western / ELISAIn Cell Western ELISA
  • 14. In Cell Western / ELISA• Coat plate with capture antibody (ELISA)• Incubate cells on plate (ICW)/grow cells (ELISA)• Fix cells (ICW)/ Add cell lysate to plate (ELISA)• Wash plates• Incubate cells with detection antibody• Wash plates• Add Fluorescent antibody (ICW)/ Add colour substrate (ELISA)• Analyze on LICOR (ICW)/ Microtiter Plate Reader (ELISA)