The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration. PubMed: 22196736
Use a concentration of 5 µg/ml. Detects a band of approximately 80 kDa (predicted molecular weight: 152 kDa).
Use at an assay dependent concentration.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Probable histone methyltransferase (By similarity). May act as a transcription regulator that binds DNA and suppresses IL5 transcription.
Involvement in disease
Note=A chromosomal aberration involving WHSC1 is a cause of multiple myeloma tumors. Translocation t(4;14)(p16.3;q32.3) with IgH. Note=WHSC1 is located in the Wolf-Hirschhorn syndrome (WHS) critical region. WHS results from by sub-telomeric deletions in the short arm of chromosome 4. WHSC1 is deleted in every case, however deletion of linked genes contributes to both the severity of the core characteristics and the presence of the additional syndromic problems.
IHC image of WHSC1/NSD2 staining in Human normal testis 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 ab75359, 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.
WHSC1/NSD2 was immunoprecipitated using 0.5mg HepG2 whole cell extract, 10µg of Mouse monoclonal to WHSC1/NSD2 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, HepG2 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 ab75359. Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution. Band: 71kDa: WHSC1/NSD2.
Western blot - Anti-WHSC1/NSD2 antibody [29D1] - ChIP Grade (ab75359)Image courtesy of an anonymous Abreview.
All lanes : Anti-WHSC1/NSD2 antibody [29D1] - ChIP Grade (ab75359) at 1/5000 dilution
Lane 1 : Whole cell lysate prepared from HeLa cells Lane 2 : Whole cell lysate prepared from HCT116 cells
Lysates/proteins at 50 µg per lane.
Secondary All lanes : HRP conjugated goat anti-mouse polyclonal at 1/10000 dilution
ab75359 (1µg/ml) staining WHSC in human testis using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining. Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1/ in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.