The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 1 µg/ml. Detects a band of approximately 72.6, 161 kDa (predicted molecular weight: 72.6 , 161 kDa).
1/50 - 1/100. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.
Histone methyltransferase. Preferentially methylates 'Lys-4' and 'Lys-27' of histone H3. H3 'Lys-4' methylation represents a specific tag for epigenetic transcriptional activation, while 'Lys-27' is a mark for transcriptional repression.
Highly expressed in brain, heart and skeletal muscle. Expressed at lower level in liver and lung.
Defects in WHSC1L1 may be involved in non small cell lung carcinomas (NSCLC). Amplified or overexpressed in NSCLC. A chromosomal aberration involving WHSC1L1 is found in childhood acute myeloid leukemia. Translocation t(8;11)(p11.2;p15) with NUP98.
Belongs to the class V-like SAM-binding methyltransferase superfamily. Histone-lysine methyltransferase family. SET2 subfamily. Contains 1 AWS domain. Contains 4 PHD-type zinc fingers. Contains 1 post-SET domain. Contains 2 PWWP domains. Contains 1 SET domain.
Breast carcinoma tissue was fixed in 10% formalin at 4°C overnight and paraffin embedded. Following blocking the tissue sections were incubated with ab4514 (1/50 dilution) at 37°C for 1 hour, and with the secondary antibody at 37°C for 30 minutes. The slides were counterstained with Hematoxylin. Washes were carried out using TBS. Heat mediated antigen retrieval was carried out using the Microwave method before commencing the staining protocol. ab4514 staining appears to be localized mainly to the cytoplasm. Although the putative cellular localization of NSD3 is the nucleus we have found no evidence in the current literature to confirm this and we believe ab4514 may be detecting a cytoplasmic form of the protein. We would be very grateful for any customer feedback about NSD3 and ab4514.
Western blot - NSD3 antibody (ab4514)
All lanes : Anti-NSD3 antibody (ab4514) at 1 µg/ml
Lane 1 : HeLa whole cell extract Lane 2 : HEK293 cell extract Lane 3 : HeLa whole cell extract with Human NSD3 peptide (ab14995) at 1 µg/ml Lane 4 : HEK293 cell extract with Human NSD3 peptide (ab14995) at 1 µg/ml
ab4514 specifically recognises NSD3 (lane1) in HeLa whole cell and HEK293 (lane3) cell extracts at 72.6 kDa and 161 kDa, which correspond to two different isoforms of NSD3. The signal can be quenched using the immunising peptide (lanes3-4) indicating that ab4514 specifically recognises NSD3.
ICC/IF image of ab4514 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab4514, 5µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).
As seen in Immunohistochemical analysis (shown above), although the putative cellular localization of NSD3 is the nucleus we have found no evidence in the current literature to confirm this and we believe ab4514 may be detecting a cytoplasmic form of the protein.
Rahman S et al. The Brd4 extraterminal domain confers transcription activation independent of pTEFb by recruiting multiple proteins, including NSD3. Mol Cell Biol31:2641-52 (2011).
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