Technical support training cell signaling
- 1. Technical training CST Michiel van der Sanden
- 2. Signaaltransductie Effector1 Kinase Phosphospecific antibody Phosphorylering Effector 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 Phosphorylering Substrate 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 inhibitor p-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/ Immunofluorescence Immunohistochemistry Immunofluorescence
- 9. Immunohistochemistry/ Immunofluorescence Immunohistochemistry 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 cytometry Grote 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 / ELISA In 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)