All lanes : Anti-Synaptophysin antibody [EP1098Y] (ab52636) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate at 20 µg
Lane 2 : SYP knockout HEK-293T cell lysate at 20 µg
Lane 3 : Human brain tissue lysate

Performed under reducing conditions.

Predicted band size: 34 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab52636 observed at 38 kDa. Red - loading control, ab7291 observed at 50 kDa.

ab52636 Anti-Synaptophysin antibody [EP1098Y] was shown to specifically react with Synaptophysin in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab267272 (knockout cell lysate ab257060) was used. Wild-type and Synaptophysin knockout samples were subjected to SDS-PAGE. ab52636 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti- Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti- Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-Synaptophysin antibody [EP1098Y] (ab52636) at 0.203 µg/ml

Lane 1 : Human brain tissue lysate
Lane 2 : Mouse brain tissue lysate
Lane 3 : Rat brain lysate tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 0.05 µg

Predicted band size: 34 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?



Blocking/Diluting buffer and concentration: 5% NFDM /TBST

PC12 cells labelled with ab52636 at 1/250 - 1/500 dilution

Anti-Synaptophysin antibody [EP1098Y] (ab52636) at 1/200000 dilution + Rat brain membrane

Secondary
Goat anti-rabbit HRP labeled at 1/2000 dilution

Predicted band size: 34 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Human medulloblastoma labelled with ab52636 at 1/250 - 1/500 dilution

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Overlay histogram showing PC12 cells stained with ab52636 (red line). The cells were fixed with methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab52636, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (0.5µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a slightly decreased signal in PC12 cells fixed with 4% paraformaldehyde (10 min) used under the same conditions.

Please note that Abcam do not have data for use of this antibody on non-fixed cells. We welcome any customer feedback.