The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use 1µg for 106 cells. ab170192-Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use a concentration of 0.5 - 1 µg/ml.
Use a concentration of 1 µg/ml. Detects a band of approximately 60 kDa (predicted molecular weight: 49 kDa).
Use at an assay dependent concentration.
FunctionParticipates in the regulation of gene transcription. Binds DNA in a non-specific manner, yet also specifically recognizes the core sequence 5'-CAC[GA]TG-3'. Seems to activate the transcription of growth-related genes.
Involvement in diseaseNote=Overexpression of MYC is implicated in the etiology of a variety of hematopoietic tumors. Note=A chromosomal aberration involving MYC may be a cause of a form of B-cell chronic lymphocytic leukemia. Translocation t(8;12)(q24;q22) with BTG1. Defects in MYC are a cause of Burkitt lymphoma (BL) [MIM:113970]. A form of undifferentiated malignant lymphoma commonly manifested as a large osteolytic lesion in the jaw or as an abdominal mass. Note=Chromosomal aberrations involving MYC are usually found in Burkitt lymphoma. Translocations t(8;14), t(8;22) or t(2;8) which juxtapose MYC to one of the heavy or light chain immunoglobulin gene loci.
Post-translational modificationsPhosphorylated by PRKDC. Phosphorylation at Thr-58 and Ser-62 by GSK3 is required for ubiquitination and degradation by the proteasome. Phosphorylation at Ser-329 by PIM2 leads to the stabilization of MYC (By similarity). Phosphorylation at Ser-62 by CDK2 prevents Ras-induced senescence. Ubiquitinated by the SCF(FBXW7) complex when phosphorylated at Thr-58 and Ser-62, leading to its degradation by the proteasome. In the nucleoplasm, ubiquitination is counteracted by USP28, which interacts with isoform 1 of FBXW7 (FBW7alpha), leading to its deubiquitination and preventing degradation. In the nucleolus, however, ubiquitination is not counteracted by USP28, due to the lack of interaction between isoform 4 of FBXW7 (FBW7gamma) and USP28, explaining the selective MYC degradation in the nucleolus. Also polyubiquitinated by the DCX(TRUSS) complex.
ab78318 staining c-Myc (phospho S62) in HeLa cells from Human Cervical Cancer by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% paraformaldehyde, permeabilized with Triton X-100 0.25% in PBS and blocked with 1.5% BSA for 30 minutes at 21°C. Samples were incubated with primary antibody (1/2000 in PBS + 1% BSA) overnight at 4°C. An Alexa Fluor®488-conjugated Goat anti-mouse IgG polyclonal(1/1000 in 1% BSA) was used as the secondary antibody incudated for 1 hour at room temperature. DAPI was used to stain the nuclear DNA.
Western blot - c-Myc (phospho S62) antibody [33A12E10] (ab78318)
All lanes : Anti-c-Myc (phospho S62) antibody [33A12E10] (ab78318) at 1 µg/ml
Lane 1 : Crude cell extracts of AGS (gastric adenocarcinoma) cells with Scr (scrambled) siRNA introduced into the cells as a negative control Lane 2 : Crude cell extracts of AGS (gastric adenocarcinoma) cells transfected with a negative control siRNA Lane 3 : Crude cell extracts of AGS (gastric adenocarcinoma) cells transfected with siRNA for c-Myc
IHC image of ab78318 staining in human normal cervical carcinoma 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 ab78318, 1µg/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.
Overlay histogram showing HL60 cells stained with ab78318 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab78318, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (IgG; H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HL60 cells fixed in 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
References for Anti-c-Myc (phospho S62) antibody [33A12E10] (ab78318)
This product has been referenced in:
Cianfanelli V et al. AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation. Nat Cell Biol17:20-30 (2015).
Read more (PubMed: 25438055) »
Ischenko I et al. Analysis of the tumor-initiating and metastatic capacity of PDX1-positive cells from the adult pancreas. Proc Natl Acad Sci U S A111:3466-71 (2014).
Read more (PubMed: 24550494) »