Cell signalling: How to easily detect mono- and poly-Ub target proteins

Recently, a new series of ready-to-use reagents for studying post-translation modifications (PTMs) of your proteins of interest have been released by Cytoskeleton, Inc. To illustrate the advantages of these research tools, this post is focuses on ubiquitination detection, which is known to be an important event in cell signalling.

Cytoskeleton Inc.’s Affinity beads (Cat. nr UBA01-beads) are optimized ubiquitin binding domain affinity reagents that effectively capture endogenous mono- and poly-Ubiquitinated target proteins. Interestingly, these Ubiquitin (Ub) affinity beads do not have heavy and light chain contamination, leading to simple and clear data interpretation as seen in the image below.

Cell signalling with Ub Affinity beads – Example with Ub-Rac1

Mono- and Poly-Ubiquitinated target proteins detected with Cytoskeleton's Signal Seeker Kits

Cell signalling studies: Mono- and Poly-Ubiquitinated target proteins detected with Cytoskeleton’s Signal Seeker Kits: 3T3 cells pretreated with MG-132 and +/- cytosolic necrotizing factor 1 (CN04) were lysed with BlastR lysis buffer. 300ug of lysate was incubated with the specified amount of the various ubiquitin affinity beads. Immunoprecipitated samples were separated by SDS-PAGE and transferred to PVDF. Western blot was performed with a Rac1 antibody. The lysis system (Cat. nr BLR01), Ub affinity beads (Cat. nr UBA01-beads), and inhibitors are all components of the Ub detection kit (Cat. nr BK161).

 

> For more information about Ub detection tools, see our ubiquitination white paper here.

Other examples of cell signalling ubiquitination regulatory mechanism identified with the Signal-Seeker Ub Detection Kit

UB Affinity beads have numerous other applications as illustrated by the detection of PD-L1 Ub in serum-restricted A431 cells pretreated with Gefitnib (see image below, or access Dr Horita’s publication: Horita H. et al. “Identifying Regulatory Posttranslational Modifications of PD-L1: A Focus on Monoubiquitinaton” Neoplasia (2017) 19, 346–353).

Signal-Seeker Ub kit for the detection of PD-L1Ub

Cell signalling studies: Serum-restricted A431 cells were pretreated with Gefitnib for 30 minutes prior to stimulation with EGF for one hour. Cells were lysed and incubated with UBA01 ubiquitin affinity beads. Samples were separated by SDS-PAGE and analyzed by western blot for PD-L1.

 

Additional uses include :

  • Investigating transient regulatory mechanisms involved in cell signalling
  • Measuring cell signalling events of multiple pathway member proteins
  • Discovering new modifications of your protein of interest
  • Gaining insight into regulatory mechanisms
  • Measuring endogenous or transiently expressed protein and cell signalling events

Related products you might also like for your cell signalling research:

    • Ubiquitination Affinity Beads (Cat. nr UBA01-beads)
    • Ubiquitin Mouse Monoclonal Antibody  (Cat. nr AUB01)
    • BlastR Rapid Lysate Prep Kit (Cat. nr BLR01)
    • Signal-Seeker Ubiquitination Detection Kits (30 Assays – Cat. nr BK161)

The Signal-Seeker products allow simple analysis of key regulatory PTMs (Acetyl-Lysine, Phosphotyrosine, SUMOylation and Ubiquitination) via affinity bead systems to isolate and enrich modified proteins from any given cell or tissue lysates. The enriched protein population is then analyzed by standard western blot procedures using a primary antibody to the target protein.

To find out more about Signal-Seekers kits, take a look at Cytoskeleton’s video!

Philippe Fixe, PhD
Written by Philippe Fixe, PhD
Philippe Fixe is Marketing Director at tebu-bio, passionate about innovation and R&D in Life sciences, Biotechnology, Medical research, Drug discovery, and also a keen photographer.