All lanes : Anti-Werner's syndrome helicase WRN antibody [195C] (ab241545) at 1 µg/ml

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : WRN knockout HAP1 cell lysate
Lane 3 : MOLT-4 cell lysate
Lane 4 : K562 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 162 kDa
Observed band size: 170 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab241545 observed at 170 kDa. Red - loading control, ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37kDa.

ab241545 was shown to react with Werner's syndrome helicase WRN in wild-type HAP1 cells in western blot. Loss of signal was observed when WRN knockout sample was used. Wild-type and WRN knockout HAP1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab241545 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at 1 µg/ml 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 Werner's syndrome helicase WRN staining in a section of formalin-fixed paraffin-embedded human testis seminoma* performed on a Leica BOND^™ 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 20mins. The section was then incubated with ab241545, 1μg/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. The inset secondary-only control image is taken from an identical assay without primary antibody.

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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

ab241545 staining Werner's syndrome helicase WRN in A431 cells. The cells were fixed with 4% PFA (10mins), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Triton for 1h. The cells were then incubated overnight at +4°C with ab241545 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^®488) at 1/1000 dilution (shown in green) and ab150084, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^®594) at 1/1000 dilution (shown in pseudocolor red). Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).