Technical training CST        Michiel van der Sanden
Signaaltransductie               Effector1                Kinase                  Phosphospecific antibody               P...
CST Produkten• Kinases and Kinase Assays• SiRNA kits• Antibodies
SiRNA kit              X             Kinase               Phosphospecific antibody            PhosphoryleringSubstrate    ...
Toepassingen phosphospecifieke          en totaal antibodies•   Western Blot (WB)•   Immunohistochemistry (IHC)•   Immunof...
Western blot         Classical HRP WB                 LICOR Fluorescent WB         -   +   SP600125 JNK inhibitorp-ATF Actin
Western blot protocol•   Cell/Tissue sample preparation in buffer•   SDS PAGE Gel•   Transfer of proteins from Gel to Blot...
Immunohistochemistry/    ImmunofluorescenceImmunohistochemistry   Immunofluorescence
Immunohistochemistry/       ImmunofluorescenceImmunohistochemistry   Immunofluorescence
IHC/IF blot protocol•   Fixation of samples•   Deparaffinization of slides (paraffin sections)•   Antigen unmasking (paraf...
Flow cytometryGrote Voordeel Flow: Single cell measurement + quantificatie
Flow blot protocol• Collection of samples• Fixation of cells• Permeabilization of cells• Aliquot and incubate cells in inc...
In Cell Western / ELISAIn Cell Western      ELISA
In Cell Western / ELISA• Coat plate with capture antibody (ELISA)• Incubate cells on plate (ICW)/grow cells (ELISA)• Fix c...
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Technical support training cell signaling

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Technical support training cell signaling

  1. 1. Technical training CST Michiel van der Sanden
  2. 2. Signaaltransductie Effector1 Kinase Phosphospecific antibody PhosphoryleringEffector 3 P Effector 3 Inactief Actief y ibod nt Dephosphorylering la ta To Phosphatase EFFECT
  3. 3. CST Produkten• Kinases and Kinase Assays• SiRNA kits• Antibodies
  4. 4. SiRNA kit X Kinase Phosphospecific antibody PhosphoryleringSubstrate P Substraat Inactief actief y ibod ant t al To
  5. 5. Toepassingen phosphospecifieke en totaal antibodies• Western Blot (WB)• Immunohistochemistry (IHC)• Immunofluorescentie (IF)• Flow cytometry (F)• Immunoprecipitatie (IP)• In Cell Western (ICW)• ELISA
  6. 6. Western blot Classical HRP WB LICOR Fluorescent WB - + SP600125 JNK inhibitorp-ATF Actin
  7. 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. 8. Immunohistochemistry/ ImmunofluorescenceImmunohistochemistry Immunofluorescence
  9. 9. Immunohistochemistry/ ImmunofluorescenceImmunohistochemistry Immunofluorescence
  10. 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. 11. Flow cytometryGrote Voordeel Flow: Single cell measurement + quantificatie
  12. 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. 13. In Cell Western / ELISAIn Cell Western ELISA
  14. 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)
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