All lanes : Anti-APE1 antibody [13B8E5C2] (ab194) at 1/2000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : APEX1 knockout HAP1 whole cell lysate
Lane 3 : HepG2 whole cell lysate
Lane 4 : HEK293 whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 35.6 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab194 observed at 37 kDa. Red - loading control, ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55kDa.

ab194 was shown to react with APEX1 in HAP1 wild-type cells in Western blot. Loss of signal was observed when APEX1 knockout sample was used. HAP1 wild-type and APEX1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween^®) before incubation with ab194 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were developed 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.

Ab194 staining Human normal placenta. Staining is localized to the nucleus.
Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS), then incubated with primary antibody for 20 minutes, 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 we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

Anti-APE1 antibody [13B8E5C2] (ab194) at 1/2000 dilution + Human fibroblast at 30 µg with Milk at 5 %

Secondary
ECL Mouse IgG,HRP - linked whole antibody (sheep) at 1/5000 dilution

Developed using the ECL technique.

Predicted band size: 35.6 kDa


Exposure time: 10 seconds

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