Detection with Ubiquitin affinity matrices

TUBEs: A Revolution in Polyubiquitin Isolation and Characterization

Traditional strategies leading to the characterization of ubiquitylated proteins often require immunoprecipitation of over-expressed Ubiquitin with an epitope tag or, the use of Ubiquitin antibodies (this latter being expensive for large scale studies). Alternatively, isolation of polyubiquitylated proteins can be achieved with certain Ubiquitin Binding Associated domains (UBAs) which nevertheless display rather low affinity for Ubiquitin. Additionally, these strategies require the inclusion of inhibitors of both Deubiquinating enzymes (DUBs) and Proteasome activity to protect the integrity of poly-ubiquitylated proteins.

These conditions could alter cell physiology, which in turn may negatively impact the result or introduce
experimental artifact.

To overcome these problems, Tandem Ubiquitin Binding Entities (TUBEs) were developed (Life Sensors).
TUBEs are essentially  tandem UBAs with dissociation constants for tetra-ubiquitin in the nanomolar range. TUBEs have also been demonstrated to protect proteins from both deubiquitylation and proteasome-mediated degradation, even in the absence of inhibitors normally required to block such activity. The nanomolar affinity of TUBEs for poly-ubiquitylated proteins allows the efficient isolation and identification of ubiquitylated proteins
from cells, tissues and organs
.

Why using TUBEs?

For which applications?


Guidelines for choosing the TUBEs to match your needs

       Reagents         

       References         

Applications

Affinity tagged TUBEs

(GST or His)

 

                  NEW!!!!!

TUBE 1 GST
TUBE 2 GST

TUBE 1 HIS
TUBE 2 HIS
TUBE 3 HIS

Flag®K63-TUBE 1

  • identification and characterisation of polyubiquitin proteins by isolation of proteins

 

 

  • Capture and isolation of K63-linked polyubiquitin proteins from cell & tissue lysates

Agarose-TUBEs

TUBE 1 Agarose
TUBE 2 Agarose

  • identification and characterisation of polyubiquitin proteins by Western blotting, as well as isolation of proteins
  • "One-step" pull downs of polyubiquitylated proteins

Biotin-TUBEs

 

 

                 NEW!!!!!

TUBE 1 Biotin
TUBE 2 Biotin

 

K63-specific TUBE 1 Biotin

  • Replacement of anti-ubiquitin antibodies, as well as the required blot heating
  • Detection of polyubiquitinated proteins by ligand blotting ("far western blotting")
  • Highly sensitive detection of K63 polyubiquitinated chains (from 100 to 1000-fold weaker affinity for chains linked through K48 or K11)

Fluorescent-TUBEs

FLR-TUBE 2
TMR-TUBE 2

  • Cytochemical staining of polyubiquitylated proteins for fluorescence microscopy.
  • Since the fluorophore is attached to the tag, it does not affect binding of the tandem ubiquitin binding domains to polyubiquitin chains.

    FLR-TUBE2 has an average of three fluorescein moieties attached to the fusion tag of His6-TUBE2. TMR-TUBE2 has a single TAMRA fluorophore attached to the fusion tag of His6-TUBE2.

HRP-TUBEs

HRP conjugated TUBE2

  • Detection of polyubiquitylated proteins by ligand blotting.
  • In situ labeling for detection of polyubiquitin by histochemistry