All lanes : Anti-WHSC1/NSD2 antibody [29D1] (ab75359)

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : WHSC1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 152 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab75359).

  Lanes 1- 2: Merged signal (red and green). Green - ab75359 observed at 80 kDa. Red - Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) observed at 37 kDa.

 ab75359 was shown to react with WHSC1/NSD2 in wild-type HeLa cells in western blot. The band observed in knockout cell line ab264936 (knockout cell lysate ab257798) lane below 80kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and WHSC1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab75359 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at a 5 µg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^®800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^®680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

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 70^oC; 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, Azide and Arginine (ab75359).

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, Azide and Arginine (ab75359).