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)