Anti-MAP1LC3A antibody [EP1528Y] (ab52628) at 1/50000 dilution + Fetal brain lysate at 10 µg

Secondary
goat anti-rabbit HRP labelled at 1/2000 dilution

Predicted band size: 14 kDa
Observed band size: 16 kDa why is the actual band size different from the predicted?

Immunocytochemistry/ Immunofluorescence analysis of SH-SY5Y (human neuroblastoma epithelial cell) cells labeling MAP1LC3A with purified ab52628 at 1/100 dilution (10 µg/mL). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL) was used as the secondary antibody only control.

IHC image of ab52628 staining in human hippocampus 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 ab52628, 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.

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.