Your SlideShare is downloading. ×
0
Technical support training cell signaling
Technical support training cell signaling
Technical support training cell signaling
Technical support training cell signaling
Technical support training cell signaling
Technical support training cell signaling
Technical support training cell signaling
Technical support training cell signaling
Technical support training cell signaling
Technical support training cell signaling
Technical support training cell signaling
Technical support training cell signaling
Technical support training cell signaling
Technical support training cell signaling
Upcoming SlideShare
Loading in...5
×

Thanks for flagging this SlideShare!

Oops! An error has occurred.

×
Saving this for later? Get the SlideShare app to save on your phone or tablet. Read anywhere, anytime – even offline.
Text the download link to your phone
Standard text messaging rates apply

Technical support training cell signaling

153

Published on

Published in: Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
153
On Slideshare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
2
Comments
0
Likes
0
Embeds 0
No embeds

Report content
Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
No notes for slide

Transcript

  • 1. Technical training CST Michiel van der Sanden
  • 2. Signaaltransductie Effector1 Kinase Phosphospecific antibody PhosphoryleringEffector 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 PhosphoryleringSubstrate 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 inhibitorp-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/ ImmunofluorescenceImmunohistochemistry Immunofluorescence
  • 9. Immunohistochemistry/ ImmunofluorescenceImmunohistochemistry 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 cytometryGrote 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 / ELISAIn 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)

×