Anti Pan Ubiquitin Monobody (Biotin labeled)

Cosmo Bio LTD

Catalog No: CAC-MB-001-B

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Anti Pan Ubiquitin Monobody (Biotin labeled)

Application: IP, IHC, ICC, IF, ELISA, WB 
Clonality: recombinant "monobody" MW ~14.3 kDa 
Purification: Immobilized Metal Chromatography 
Reactivity: All 

This product is a single-chain artificial antibody (monobody) that recognizes ubiquitin monomer and mono/poly-ubiquitinated proteins.
It has several properties superior to existing monoclonal antibody products and is ideal for immunoprecipitation (IP) and various immunostainings (IHC/ICC/IF).

*This product is commercialized under license from the Tokyo Metropolitan Institute of Medical Science, Tokai National University Organization, and Kyoto University.

Features

• High affinity - Among the ubiquitin-binding clones screened from the monobody combinatorial library (10 ¹³ types), the clone with the highest affinity has been commercialized. Generally, IgG antibodies are considered to have high affinity with a dissociation constant (Kd) in the low nanomolar range, but this monobody was determined to have a dissociation constant (Kd) of 0.88 nM by biolayer interferometry analysis, indicating high affinity.
• High permeability - Monobodies have a molecular size less than 1/10 that of common IgG, which makes them highly permeable, similar to VHH antibodies (nanobodies). In immune cell staining, this monobody has been shown to detect ubiquitin better than the existing mouse monoclonal antibody (FK2) (Figure 1).
• With low bias - this monobody has been shown to be able to efficiently immunoprecipitate ubiquitin monomers and monoubiquitinated histones (Figure 3), which is superior to existing mouse monoclonal antibodies (FK2, P4D1). . It is also excellent for immunoprecipitation of polyubiquitinated proteins, and all eight ubiquitin chain types (K6, K11, K27, K29, K33, K48, K63, M1) can be efficiently detected by quantitative mass spectrometry analysis of immunoprecipitated proteins. It has been shown that it can be recovered at a rate that closely matches that in the cell extract (Figure 4). This suggests that this monobody binds to all ubiquitin chain types with a low bias, and we speculate that the small molecular size also contributes to this property.
• Artificial antibody -monobody is an artificial antibody based on a structure derived from fibronectin type III domain (FN3). This product is C-terminally fused with a His tag and manufactured under animal-free conditions using an E. coli expression system and affinity purification. The C-terminus is also labeled with biotin, and the use of an avidin/streptavidin reagent is recommended for detection and immobilization of this product. Please refer to the related products below .

Verified applications

• Immunoprecipitation (IP)
• Immunocytochemistry (ICC)/IF: Immunofluorescence (IF)
• Immunohistochemistry (IHC)
• Western blot (WB)
• ELISA

* Monobodies have high specific activity, and better results are obtained at higher dilutions (lower concentrations) than when using general antibody products.

Example of use

Immune cell staining

Figure 1

HCT-116 cells stably expressing EGFP-ubiquitin fusion protein were treated with puromycin to induce the formation of ubiquitin-positive aggregates, and then treated with TAMRA-labeled ubiquitin monobody (A) or mouse monoclonal antibody (FK2) and Alexa568-labeled secondary antibody. Immunocyte staining was performed using the following antibody (B).
Data provided by the Tokyo Metropolitan Institute of Medical Science

Western blot

Figure 2 Detection of ubiquitinated proteins by Western blotting

HCT-116 cell lysate treated with drug (TAK-243/Btz/none) was tested using this product: Anti-Pan Ubiquitin Monobody (left) and Anti-Pan Ubiquitin Mouse IgG, Western blotting was performed using clone P4D1 (right) and visualized using HRP and chemiluminescence systems. When this product was used, signals from monoubiquitinated histone H2A and small mono/poly-ubiquitinated proteins were detected more strongly than when Mouse IgG, clone P4D1 was used. On the other hand, Western blotting showed that this product does not recognize ubiquitin monomers.
*This product recognizes ubiquitin monomer in ELISA (Figure 6) and immunoprecipitation (Figure 3).

Immunoprecipitation

Figure 3.

Immunoprecipitation of HCT-116 cell extracts was performed using various monobodies and mouse IgG, and Western blot analysis was performed using a mouse monoclonal antibody (P4D1). Only ubiquitin monobodies were able to immunoprecipitate ubiquitin monomers and monoubiquitinated histones.

Quantitative mass spectrometry of immunoprecipitates

Figure 4
Immunoprecipitates of 62 kDa or higher in Figure 3 were subjected to quantitative mass spectrometry using LC-MS/MS.

Quality Check

Figure 5 SDS-PAGE
(1) 1 µg monobody (this product), (2) 1 µg monobody incubated with 5 µg avidin, (3) 5 µg avidin separated by SDS-PAGE, then gel was stained with CBB. It was confirmed that the monobody was highly pure and biotin-labeled in high yield.

Figure 6:

A dilution series of this product (0.5 mg/mL) was reacted on an immunoassay plate (1 µg/well) coated with recombinant human ubiquitin monomer, followed by secondary reaction with NeutrAvidin-HRP, color development with TMB substrate, The reaction was stopped with sulfuric acid. It was confirmed that this product binds to recombinant human ubiquitin monomer and has a high potency.