The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use a concentration of 1 µg/ml. Detects a band of approximately 75 kDa (predicted molecular weight: 68 kDa).
Use 5 µg for 25 µg of chromatin.
Use a concentration of 1 µg/ml.
Essential component of a MLL/SET1 histone methyltransferase (HMT) complex, a complex that specifically methylates 'Lys-4' of histone H3 (H3K4). Functions as a transcriptional regulator. Binds to the TERT promoter and represses telomerase expression. Plays a role in TGFB1-mediated inhibition of cell-proliferation, possibly regulating SMAD3 transcriptional activity. Represses JUND-mediated transcriptional activation on AP1 sites, as well as that mediated by NFKB subunit RELA. Positively regulates HOXC8 and HOXC6 gene expression. May be involved in normal hematopoiesis through the activation of HOXA9 expression (By similarity). May be involved in DNA repair.
Involvement in disease
Defects in MEN1 are the cause of familial multiple endocrine neoplasia type I (MEN1) [MIM:131100]. Autosomal dominant disorder characterized by tumors of the parathyroid glands, gastro-intestinal endocrine tissue, the anterior pituitary and other tissues. Cutaneous lesions and nervous-tissue tumors can exist. Prognosis in MEN1 patients is related to hormonal hypersecretion by tumors, such as hypergastrinemia causing severe peptic ulcer disease (Zollinger-Ellison syndrome, ZES), primary hyperparathyroidism, and acute forms of hyperinsulinemia. Defects in MEN1 are the cause of familial isolated hyperparathyroidism (FIHP) [MIM:145000]; also known as hyperparathyroidism type 1 (HRPT1). FIHP is an autosomal dominant disorder characterized by hypercalcemia, elevated parathyroid hormone (PTH) levels, and uniglandular or multiglandular parathyroid tumors.
Phosphorylated upon DNA damage, probably by ATM or ATR.
Nucleus. Concentrated in nuclear body-like structures. Relocates to the nuclear matrix upon gamma irradiation.
Western blot - Anti-Menin antibody - ChIP Grade (ab31902)
Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: Menin knockout HAP1 cell lysate (20 µg) Lane 3: Jurkat cell lysate (20 µg) Lane 4: A431 cell lysate (20 µg) Lanes 1 - 4: Merged signal (red and green). Green - ab31902 observed at 74 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab31902 was shown to recognize Menin when Menin knockout samples were used, along with additional cross-reactive bands. Wild-type and Menin knockout samples were subjected to SDS-PAGE. ab31902 and ab8245 (loading control to GAPDH) were diluted 1 µg/mL and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.
Western blot - Menin antibody (ab31902)
All lanes : Anti-Menin antibody - ChIP Grade (ab31902) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 :Jurkat whole cell lysate (ab7899) Lane 3 :A431 whole cell lysate (ab7909) Lane 4 :HEK293 whole cell lysate (ab7902)
Lysates/proteins at 10 µg per lane.
Secondary All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution
Menin was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Menin and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab31902. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 75kDa: Menin.
ChIP - Anti-Menin antibody - ChIP Grade (ab31902)
ChIP analysis of Menin along the PCNA promoter. High enrichment at PCNA PRO-2 and weak enrichment at PCNA PRO-1 is observed as previously described in literature.
Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab31902 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach). Primers are located in the first kb of the transcribed region.
ICC/IF image of ab31902 stained human HEK 293 cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab31902, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
ICC/IF image of ab31902 stained MCF7 cells. The cells were 4% PFA fixed (10 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 (ab31902, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit 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.