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Monoclonal Antibodies

Anti–BirA [5B11c3-3]

Invented by Brian Burke
Invented at A*STAR Accelerate Technologies Pte Ltd
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Western blot analysis of 5B11c3-3 monoclonal antibody. Immortalized human fibroblast (HF) (control) or HF stably expressing a green fluorescence protein (GFP) fused to a nuclear localization signal (NLS) and BirA (GFP-NLS-BirA) without (- dox) or with (+ dox) induced expression were lysed and analysed by Western blot for BirA with 5B11c3-3 antibody. Both primary and secondary antibodies were diluted in blocking buffer (5% nonfat dry milk resuspended in 0.1% Tween and 1X PBS). The predicted GFP-NLS-BirA molecular weight is 66 kDa.
Confocal images of cells expressing BirA-tagged constructs. (A) Human fibroblasts (HF) stably expressing green fluorescence protein (GFP) fused to a nuclear localization signal (NLS) and BirA (GFP-NLS-BirA), (B) HEK293 stably expressing BirA tagged to a nuclear envelope protein SLAP75 (BirA-SLAP75) and (C) Hela cells transfected with red fluorescent protein fused to BirA amino acid residues 151-321 (RFP-BirA 151-321) were fixed in 4% PFA (A, C) or cold methanol (B) and immunofluorescently stained for BirA with 5B11c3-3 monoclonal antibody. The nuclei were visualised with Hoechst.
The anti-BirA antibody clone 5B11c3-3 recognized miniTurbo BirA variant. Immortalized human fibroblast (HF) stably expressing miniTurbo without (- dox) or with (+ dox) induced expression were lysed and analysed by Western blot with the 5B11c3-3 antibody. Both primary and secondary antibodies were diluted in blocking buffer (5% nonfat dry milk resuspended in 0.1% Tween and 1X PBS). The predicted miniTurbo molecular weight is 28 kDa.
Confocal images of human fibroblasts (HF) stably expressing miniTurbo. HF were fixed in 4% PFA and immunofluorescently stained for miniTurbo with 5B11c3-3 monoclonal antibody. The nuclei were counter-stained with Hoechst.
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Catalogue Number 153487
Applications IF WB
Antigen/Gene or Protein Targets BirA R118G
Synonyms BirA antibody, bioR antibody, dhbB antibody, JW3941 antibody, BirA miniTurbo antibody, BirA/miniTurbo
Reactivity E.coli
Relevance Monoclonal antibody which specifically detects BirA in E.coli samples.

Background and Research Application
E.coli BirA is a well-characterized multifunctional protein that either catalyses the transfer of biotin to biotin carboxyl carrier protein (BCCP), an acetyl-CoA carboxylase subunit or, alternatively, binds to the biotin operator resulting in transcriptional repression of the biotin operon. This protein also activates biotin to form biotinyl-5'-adenylate and transfers the biotin moiety to biotin-accepting proteins. A mutant BirA (R118G) shows loss of DNA binding ability and promiscuity of substrate biotinylation. These properties of BirA R118G were used to develop an in vivo proximity labelling technique that identifies potential interacting proteins (BioID). In the BioID method, a protein of interest is tagged with BirA R118G and expressed in live cells. Addition of the biotin to the culture medium results in biotinylation of proteins in vicinity of the bait. The biotinylated proteins can then be affinity purified with biotin binding proteins such as streptavidin or avidin even under harsh denaturing conditions and subsequently identified by mass spectrometry or immunoblot analysis.

One of the drawbacks of the BirA/BioID is its slow biotinylation kinetics making it unsuitable for the study of dynamic processes. To overcome the hurdle directed evolution of BirA R118G was used to generate two BirA variants with superior enzymatic activity: (i) 35 kD TurboID, with 15 mutations relative to wild-type BirA; and 28 kD miniTurbo, with N-terminal domain deleted and 13 mutations relative to the original BirA. This antibody specifically detects BirA in E. coli samples.
Host Mouse
Immunogen Glutathione-S-transferase (GST) fused to E.coli BirA R118G
Immunogen UniProt ID P06709
Positive Control Cells overexpressing an E.coli BirA/miniTurbo construct
Subclass IgG1 kappa
Molecular Weight (kDa) 35
Myeloma Used Sp2/0-Ag14
Strain C57BL/6
Notes Production Details
Purified using multi-step affinity chromatography with protein A.

Storage Conditions
Store at -20 degrees frozen. Avoid repeated freeze/thaw cycles.

Points of Interest
Specificity:
The antibody recognises the C-terminal half of E.coli BirA; the antibody recognised the BirA variant miniTurbo; not tested for TurboID or other BirA derivate

Epitope: Amino acids residues 151-321 of E.coli BirA

Entrez Gene Number: 948469
Gene Symbol: birA, bioR, dhbB, JW3941

Concentration
1mg/ml as standard
Research Area Epitope Tags

References

There are 6 reference entries for this reagent.

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References: 6 entries

Creed et al. 2020. Development. 147(1):. PMID: 31806659.

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Branon et al. 2018. Nat Biotechnol. 36(9):880-887. PMID: 30125270.

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Roux et al. 2012. J Cell Biol. 196(6):801-10. PMID: 22412018.

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Choi-Rhee et al. 2004. Protein Sci. 13(11):3043-50. PMID: 15459338.

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Streaker et al. 2003. J Mol Biol. 325(5):937-48. PMID: 12527300.

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Kwon et al. 2000. J Mol Biol. 304(5):821-33. PMID: 11124029.

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References: 6 entries

Creed et al. 2020. Development. 147(1):. PMID: 31806659.

View  

Branon et al. 2018. Nat Biotechnol. 36(9):880-887. PMID: 30125270.

View  

Roux et al. 2012. J Cell Biol. 196(6):801-10. PMID: 22412018.

View  

Choi-Rhee et al. 2004. Protein Sci. 13(11):3043-50. PMID: 15459338.

View  

Streaker et al. 2003. J Mol Biol. 325(5):937-48. PMID: 12527300.

View  

Kwon et al. 2000. J Mol Biol. 304(5):821-33. PMID: 11124029.

View  

Add a reference

Inventor Information