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October 29, 2015

Easy quantification of DUB activity

 

fluorescent ubiquitin probes 

 

Immunoblotting procedures using ubiquitin probes with an affinity tag are widely used to monitor DUB inhibition in DUB inhibitor potency and specificity profiling studies.

Here we would like to make a plea for in-gel fluorescent scanning by using fluorescently labeled  probes. A method with many advantages:

fluorescent activity based  DUB probes

  • time saving (direct read-out <1 h)
  • reliable, reproducible quantification of DUB activity
  • sensitive and high resolution
  • a large lineair window for quantifications
  • no background labelling due to antibody
 

application: to quantify or visualise DUB activity in response of chemical or genetical modulation on the protein level.

immunoblotting vs. fluorescence scanning

 

When you compare a western blot (HA-Ub-VME) with an in-gel fluorescence scan (TAMRA-Ub-VME) the differences are obvious:

Application: IC50 determination of DUB inhibition

 

One of the main advantages of fluorescent probes is that they enable quantitative assessment of genetic or chemical inhibition of DUB activity.

In the example below the IC50 is determined for a specific DUB of the small-molecule DUB inhibitor (b-AP15), using our TAMRA-Ub-VME probe (UbiQ-050).

This can be done for any DUB activity in a complex lysate! A great tool for analysing DUB specificity of your inhibitor in cellular context.
 

order your fluorescent probes today:

 

UbiQ-050TAMRA-Ub-VME 
UbiQ-058 : TAMRA-Ub-PA (Prg)
UbiQ-071 : 
Cy5-Ub-VME
UbiQ-072 : Cy5-Ub-PA (Prg)

view our full catalogue

Reference 


All images and data were kindly provided by dr Annemieke de Jong. These images and more information and can also be found in the following article: 
[de Jong, A., Merkx, R., Berlin, I., Rodenko, B., Wijdeven, R. H. M., El Atmioui, D., Yalçin, Z., Robson, C. N., Neefjes, J. J. and Ovaa, H. (2012), Ubiquitin-Based Probes Prepared by Total Synthesis To Profile the Activity of Deubiquitinating Enzymes. ChemBioChem, 13: 2251–2258.]
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