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Technical training CST
Michiel van der Sanden
Signaaltransductie
Effector1
Kinase
Effector 3Actief
Effector 3Inactief
Phosphorylering
Phosphatase
Dephosphorylering
P
EFFECT
Phosphospecific antibody Total a
ntibody
CST Produkten
• Kinases and Kinase Assays
• SiRNA kits
• Antibodies
Kinase
Substraat actief
SubstrateInactief
Phosphorylering
P
Phosphospecific antibody Total a
ntibody
X
SiRNA kit
Toepassingen phosphospecifieke en totaal antibodies
• Western Blot (WB)
• Immunohistochemistry (IHC)
• Immunofluorescentie (IF)
• Flow cytometry (F)
• Immunoprecipitatie (IP)
• In Cell Western (ICW)
• ELISA
Western blot
Actin
p-ATF
Actin
SP600125 JNK inhibitor
Actin
-
p-ATF
Actin
SP600125 JNK inhibitor+
Classical HRP WB LICOR Fluorescent WB
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
Immunohistochemistry/Immunofluorescence
ImmunofluorescenceImmunohistochemistry
Immunohistochemistry/Immunofluorescence
ImmunofluorescenceImmunohistochemistry
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
Flow cytometry
Grote Voordeel Flow: Single cell measurement + quantificatie
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
In Cell Western / ELISA
In Cell Western ELISA
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)