3 monoclonals to discriminate free Ubiquitin from linear and polyubiquitin chains

Ubiquitin (Ub) is an 8 kDa  highly conserved polypeptide, commonly expressed in eukaryotic cells. Ub is added to lysine residues of the target proteins. This post-translational modification (known as ubiquitination) is made through the sequential action of 3 enzymes (E1 Ub activating enzyme, E2 conjugating enzyme and E3 ligase).

In parallel, the 7 lysines of the Ub itself (K6, K11, K27, K29, K33, K48, and K63) are also Ub acceptors. This enables the formation of polyubiquitin chains among which, K48 and K63 linkages are the most studied.  In addition to these 7 lysines, polyubiquitin linkages can be formed through a methionine residue present in the Ub polypeptide by the Linear Ubiquitin Chain Assembly Complex (LUBAC).

Ubiquination, a post-translational modification regulating various cell functions

Like phosphorylation or sumoylation, ubiquitination controls the activity and fate of the ubiquinated proteins and thus cellular functions. It is well accepted that:

  • K48 Ub-protein linkages lead to proteasomal degradation.
  • K63-Ub-protein linkages drive cell signaling, receptor endocytosis, cell trafficking and immune responses, the role of the linear and K6, K11, K27, K29, K33 Ub-protein not being completely understood.
  • The linear polyubiquitin (M1-linked polyubiquitin) plays important roles in inflammatory and immune responses, by interacting with the NFKB signaling pathway.

3 monoclonals to discriminate Ub and polyubiquitin chains

Being able to distinguish the various forms of Ub present in a sample is very significant. It allows researchers to improve their knowledge of protein and cellular functions involved in the model analyzed. For such comparative studies, antibodies that recognize and distinguish linear polyubiquitin from polyubiquitylated proteins and free ubiquitin are necessary. Here, you will find my selection of highly qualified monoclonal antibodies to enable experimental approaches that will allow you to differentiate and localise the various UB forms in your samples;

#1 – VU-1 monoclonal anti-Ubiquitin

VU-1 is a monoclonal antibody that recognizes K48-, K63-, K11- chains and linear free ubiquitin.

VU-1 is ideal for Western blotting (recommended dilution 1:1,000 – 1:10,000), immunostaining (recommended dilution 1:200 – 1:500) and ELISA.

#2 – FK2 monoclonal anti-Ubiquitin

The FK2 monoclonal ubiquitin antibody (AB120) detects both mono- and K11-, K29-, K48- and K63-linked polyubiquitin but not free ubiquitin!  FK2 is suited for Western blotting (recommended dilution 1:1,000), immuno-precipitation and immunostaining applications.

#3 – LUB9 monoclonal anti-Ubiquitin

The Linear Polyubiquitin specific monoclonal LUB9 antibody (AB130) displays high specificity for linear polyubiquitin chains.

LUB9 is suitable for Western blotting of linear ubiquitin chains from tissue or cell lysates (recommended dilution 1:1,000 – 1:2,500). LUB9 specifically detects linear over K11-, K48- and K63-linked polyubiquitin.

TIPS ICON
Small molecules can be very useful to study this pathway. I have made a selection of small molecule active on the ubiquitin pathway for research and screening studies.
Feel free to suggest those you think 
should be added!

Want to discriminate linear- from poly- and free ubiquitin?

Think VU-1, FK2 and LUB9 monoclonal antibodies!

The experimental data described below speaks for itself…

VU-1 anti-ubiquitin antiboy

Sections of human brain stained with VU-1 ubiquitin monoclonal antibody visualized using DAB chromogenic substrate: A) Multiple System Atrophy, B) Parkinson’s Disease, C) Progressive Supranuclear Palsy – Source: LifeSensors

VU-1 monclonal anti-Ub

Rat primary neuron triple stained with antibodies to ubiquitin (VU-1; red), microtubule-associated protein 2 (MAP2; green), and the nuclear stain DAPI (blue). Source: LifeSensors.

Anti-Ub monoclonals

Ubiquitin immunoreactivity (FK2 and VU-1) in OLN93 cell line expressing a-synuclein. Cells were treated with 1 µM of MG132 for 2 h and fixed in methanol. Cytoplasmic a-synuclein-positive, red (SNL-4) and Ubiquitin-positive inclusions, green (FK2, 1:200; VU-1, 1:200).Images provided by Prof. Christiane Richter-Landsberg, Oldenburg University, Germany. Source: LifeSensors.

Anti-Ub Monoclonals

Ubiquitin immunostaining (FK2, 1:500; VU-1, 1:500; red) in US02 cell line after focused laser beam induced DNA damage. The presence of DNA double strand breaks are visualized using histone rabbit polyclonal antibody (1:500, yH2AX, green). Images provided by Youssef Shiloh, Ph.D., Tel Aviv University. Source: LifeSensors.

Anti-Ub monoclonals

Ubiquitin immunoreactivity in rat primary neurons (FK2, 1:500; VU-1, 1:500; red). Microtubule-associated protein 2 (MAP2) was used as a neuronal marker (1:500, green). Images provided by BethAnn McLlaughlin,Ph.D., Vanderbilt University. Source: LifeSensors.

Anti-Ub monoclonals

Ubiquitin-positive (FK2 and VU-1) and Tau-labeled neurons in human brain tissue (Progressive Supranuclear Palsy, PSP). Paraffin embedded, formalin fixed 6 μm sections were stained with MAb FK2 (1:1000) or VU-1 (1:1000) after antigen retrieval. To block autofluorescence, tissue sections were treated with Sudan black. Source: LifeSensors

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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  1. […] in our samples. It is also useful to enrich specific-protein modifications (e.g. phosphorylation, ubiquitination) in heterogeneous samples (e.g. cell lysates). In brief, the IP antibody recognises the specific […]

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