The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 52 kDa (predicted molecular weight: 49 kDa).
Use a concentration of 5 µg/ml.
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.
Lane 1 : NIH3T3 (-AP) Lane 2 : NIH3T3 (+AP) Lane 3 : NIH3T3 + C peptide with c-Myc peptide (ab116700) Lane 4 : NIH3T3 + P peptide with c-Myc (phospho T58) peptide (ab104386) Lane 5 : Raji (-AP) Lane 6 : Raji (+AP)
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size : 49 kDa
The blots were produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 55 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membranes were blocked for an hour, membrane strips 2 and 6 were incubated with alkaline phospatase (100 U per mL) and strips 1 and 5 in phosphatase reaction buffer without enzyme for one hour at 37 degrees before incubating lane 1 - 4 with ab85380 (rabbit polyclonal to c-Myc (phospho T58) antibody; 1 ug per mL) and lane 5-6 with ab32072 (rabbit monoclonal to c-Myc; 1:10000 dilution), in the presence of loading control ab8245 (mouse anti-GAPDH antibody; diluted 1:20000) for 24 hours at 4°C. Prior to adding the primary antibodies to the strips 3-4, 1 ug per mL of ab116700 (c-Myc control peptide: lane 3) or ab104386 (c-Myc (phospho T58) peptide: lane 4) were added to the antibodies.
Serially diluted ab85380 was bound to immobilised phospho (ab104386) - or control (ab116700) peptides (1 microgram x mL-1). The antibody was detected by HRP-labelled goat anti-rabbit IgG (ab97080; diluted 50000 times) and signal was developed with TMB substrate.
Western blot - c-Myc (phospho T58) antibody (ab85380)
All lanes : Anti-c-Myc (phospho T58) antibody (ab85380) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252) with c-Myc (phospho T58) peptide (ab104386) at 1 µg/ml Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252) with c-Myc peptide (ab116700) at 1 µg/ml Lane 3 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252)
Lysates/proteins at 10 µg per lane.
Secondary Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution developed using the ECL technique
ICC/IF image of ab85380 stained MCF-7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab85380 at 5µg/ml overnight at +4°C. The secondary antibody (green) was a goat anti-rabbit DyLight® 488(ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
References for Anti-c-Myc (phospho T58) antibody (ab85380)