Radiometric kinase assays with scintillation counting are a super sensitive, top notch, way to use radioactive ATP to track the phosphorylation of proteins and peptides by kinases. More on all that mumbo jumbo in the video but basically the kinase transfers the hot phosphate to the protein, you stick that protein/peptide onto a piece of phosphocellulose paper (ala P81), wash off the excess ATP, dry the paper, stick it into a liquid scintillant that will dissolve your sample, take the energy given off by the radioactive phosphate (if your protein/peptide got kinased (technical term is phosphorylated)), & give off light that will detected by a dinosaur of a machine (old and powerful so dino fits it well). 

I’m not going to write much text but I will include some great links and an overview of the protocol I use for the measuring part (the actual kinase part is totally dependent on the kinase & substrate but here’s a link to an article with more info: https://www.nature.com/articles/nprot.2006.149 ) & of course, see the Newton Lab website http://newtonlab.ucsd.edu/protocols/ 

and special thanks this week to Drs. Alexandra Newton, Nicholas Tonks, & Prabhadevi Venkataramani for helping me learn these assays and (in the case of Dr. Tonks, letting me use his equipment!)



scintillation counting experiments – basic measuring protocol

Phosphocellulose paper (P81 or equivalent): link to buying the equivalent (thanks so much Jon Oakhill at Saint Vincent’s Institute of Medical Research): https://www.svi.edu.au/resources/phosphocellulose_paper/ 

  • cut into 2x1cm pieces, label tops (with pencil!) and organize in box
    • use pencil because solvent will dissolve pen ink – eek!

scintillation vials – prepare ahead of time:

  • 2mL scintillation fluid per vial – I typically label & line all the vials up in the rack (caps off) and then fill a 10mL pipet to 12mL and transfer 2 per tube, repeat for all tubes
  • need to prepare 1 vial per sample + 3 for ATP master mix (MM) only, also make a couple extras because it’s science and mistakes happen
  • stick on caps

phosphoric acid: prepare 1 L 0.5% (75mM) phosphoric acid for washes (need 200-300mL per wash)

  • stock is 85%

(85)(x) = (0.5)(1000)

x = ~5.8mL phosphoric acid – pipet directly into water in 2 L glass bottle

remember do whatcha oughta – add acid to wata! 

scintillation counting

  • prepare phosphoric acid wash
    • ~250-300mL 0.5% phosphoric acid + magnetic stir bar in the acid beaker (enough to submerge basket)
  • insert basket
  • spot sample onto the center of P81 paper and drop into basket
    • I spot 15uL of each of the reactions but it will depend on your experiment and how hot (radioactive) it is
  • let wash for 5 min
  • stop spinning, remove basket – let drain into beaker, then transfer basket to paper in plastic tray
  • use tongs to remove stir bar
  • pour out acid into the acid waste container
  • repeat 2 X (so 3 acid washes)
  • wash 1X in acetone (~300mL) for 5 min – transfer beaker to paper on tray
  • transfer filter papers to paper to air dry (only takes a few minutes)
  • spot 2uL of the ATP MM onto a filter paper to quantify (no wash! the point of the wash is to remove it!)
  • measure
  • I set it for automatic counting, 50 seconds
  • put in scintillation racks followed by halt rack 
  • follow machine turn on instructions (you have to plug in and turn on computer/printer/machine in specific order for the one I use))
  • clean up 
  • pour 5% decon solution into beakers & stick in tweezers, tongs, pipets (so tips are in) & let soak
  • check, and dispose of
  • scan surfaces & use decon to wipe down

more on kinases: https://bit.ly/kinasesandphosphorylation 

more on radioactivity: https://bit.ly/radioactivitybiochem 

link to that scintillant brochure I showed: https://search.cosmobio.co.jp/cosmo_search_p/search_gate2/docs/icn_/880020.20061205.pdf 

link to lots of helpful stuff from PerkinElmer’s website: https://www.perkinelmer.com/lab-products-and-services/application-support-knowledgebase/radiometric/liquid-scintillation-counting.html 


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