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)