Key features and details
- Mouse monoclonal [246C5a] to CBLB
- Suitable for: IHC-P, WB
- Knockout validated
- Reacts with: Human
- Isotype: IgG2a
Product nameAnti-CBLB antibody [246C5a]
See all CBLB primary antibodies
DescriptionMouse monoclonal [246C5a] to CBLB
Tested applicationsSuitable for: IHC-P, WBmore details
Species reactivityReacts with: Human
Recombinant C terminal fragment of Human CBLB.
- WB: CTLL-2, HeLa and MCF7 cell lysates
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term.
Storage bufferpH: 7.40
Preservative: 0.05% Sodium azide
Constituents: 1% BSA, PBS
Concentration information loading...
PurityProtein G purified
Purification notesab54362 was purified using protein G column chromatography from culture supernatant of hybridoma cultured in a medium containing bovine IgG depleted (approximately 95%) fetal bovine serum and filtered through a 0.22µm membrane.
KO cell lines
KO cell lysates
Our Abpromise guarantee covers the use of ab54362 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|WB||Use at an assay dependent concentration. Predicted molecular weight: 109 kDa.|
RelevanceFunction: E3 ubiquitin-protein ligase which accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes, and transfers it to substrates, generally promoting their degradation by the proteasome. Negatively regulates TCR (T-cell receptor), BCR (B-cell receptor) and FCER1 (high affinity immunoglobulin epsilon receptor) signal transduction pathways. In naive T-cells, inhibits VAV1 activation upon TCR engagement and imposes a requirement for CD28 costimulation for proliferation and IL-2 production. Also acts by promoting PIK3R1/p85 ubiquitination, which impairs its recruitment to the TCR and subsequent activation. In activated T-cells, inhibits PLCG1 activation and calcium mobilization upon restimulation and promotes anergy. In B-cells, acts by ubiquitinating SYK and promoting its proteasomal degradation. Slightly promotes SRC ubiquitination. May be involved in EGFR ubiquitination and internalization. May be functionally coupled with the E2 ubiquitin-protein ligase UB2D3. Tissue specificity: Expressed in placenta, heart, lung, kidney, spleen, ovary and testis, as well as fetal brain and liver and hematopoietic cell lines, but not in adult brain, liver, pancreas, salivary gland, or skeletal muscle. Present in lymphocytes (at protein level). Pathway: Protein modification; protein ubiquitination. Similarity: Contains 1 Cbl-PTB (Cbl-type phosphotyrosine-binding) domain. Contains 1 RING-type zinc finger. Contains 1 UBA domain. Domain: The N-terminus is composed of the phosphotyrosine binding (PTB) domain, a short linker region and the RING-type zinc finger. The PTB domain, which is also called TKB (tyrosine kinase binding) domain, is composed of three different subdomains: a four-helix bundle (4H), a calcium-binding EF hand and a divergent SH2 domain. The RING-type zinc finger domain mediates binding to an E2 ubiquitin-conjugating enzyme. The UBA domain interacts with poly-ubiquitinated proteins. PTM: Phosphorylated on tyrosine and serine residues upon TCR or BCR activation, and upon various types of cell stimulation. Auto-ubiquitinated upon EGF-mediated cell activation or upon T-cell costimulation by CD28; which promotes proteasomal degradation.
Cellular localizationCytoplasmic and Nuclear
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All lanes : Anti-CBLB antibody [246C5a] (ab54362) at 1/500 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CBLB knockout HeLa cell lysate
Lane 3 : CTLL-2 cell lysate
Lane 4 : MCF7 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 109 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?
Lanes 1-4: Merged signal (red and green). Green - ab54362 observed at 130 kDa. Red - loading control, ab181602 observed at 37 kDa.
ab54362 Anti-CBLB antibody [246C5a] was shown to specifically react with CBLB in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264711 (knockout cell lysate ab258342) was used. Wild-type and CBLB knockout samples were subjected to SDS-PAGE. ab54362 and Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.
Anti-CBLB antibody [246C5a] (ab54362) + immunizing recombinant protein
Predicted band size: 109 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?
The molecular weight of the band on the Western blot does not correspond to the molecular weight of the natural protein as the immunizing recombinant protein fragment was used as the test antigen.
IHC image of ab54362 staining in human T-cell lymphoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab54362, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
ab54362 has been referenced in 2 publications.
- Singh TP et al. Cbl-b deficiency provides protection against UVB-induced skin damage by modulating inflammatory gene signature. Cell Death Dis 9:835 (2018). PubMed: 30082827
- Wallner S et al. The role of the e3 ligase cbl-B in murine dendritic cells. PLoS One 8:e65178 (2013). WB ; Mouse . PubMed: 23762309