All lanes : Anti-NF2 / Merlin antibody [AF1G4] (ab88957) at 1/2000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : NF2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 69 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab88957 observed at 60 kDa. Red - loading control ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37kDa.

ab88957 was shown to react with NF2 in wild-type HeLa cells in western blot with loss of signal observed in NF2 knockout cell line ab261796 (NF2 knockout cell lysate ab257179). Wild-type and NF2 knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab88957 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

IHC image of NF2/Merlin staining in human skeletal muscle formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM 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 ab88957, 10µg/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.

ICC/IF image of ab88957 stained HeLa cells. The cells were 4% formaldehyde 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 (ab88957, 1/200 dilution) overnight at +4°C. The secondary antibody (green) was ab88957, DyLight® 488 goat anti-mouse IgG (H+L) used at a 1/250 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.

Lane 1: Wild-type HAP1 cell lysate (40 µg)
Lane 2: Empty lane
Lane 3: NF2 / Merlin knockout HAP1 cell lysate (20 µg)
Lane 4: Empty lane
Lanes 1 - 4: Merged signal (red and green). Green - ab88957 observed at 70 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab88957 was shown to specifically react with NF2/Merlin in wild-type HAP1 cells. No band was observed when NF2 / Merlin knockout samples were examined. Wild-type and NF2/Merlin knockout samples were subjected to SDS-PAGE.  Ab88957 and ab181602 (loading control to GAPDH) were diluted at 1/500 and 1/10,000 dilution respectively and incubated overnight at 4^oC. Blots were developed with IRDye® 800CW Goat anti-Mouse IgG (H + L) ab216772 and IRDye® 680 Goat anti-Rabbit IgG (H + L) ab216777 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-NF2 / Merlin antibody [AF1G4] (ab88957) at 1/2000 dilution

Lane 1 : HeLa cell lysate
Lane 2 : 293T cell lysate
Lane 3 : MCF7 cell lysate
Lane 4 : HepG2 cell lysate
Lane 5 : MOLT4 cell lysate
Lane 6 : C6 cell lysate
Lane 7 : L929 cell lysate

Predicted band size: 69 kDa
Observed band size: 69 kDa