Anti-Calbindin antibody [AF2E5] (ab75524) at 1/2000 dilution (in blocking buffer for 16 hours at 4°C) + Mouse hippocampus whole tissue lysate at 5 µg

Secondary
An HRP-conjugated Horse anti-mouse IgG polyclonal at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 30 kDa
Observed band size: 25 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds


Blocking Step: 5% Milk for 1 hour at 25°C

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All lanes : Anti-Calbindin antibody [AF2E5] (ab75524) at 1/500 dilution

Lane 1 : Human kidney tissue lysate - total protein (ab30203)
Lane 2 : Kidney (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 30 kDa
Observed band size: 30 kDa


Exposure time: 90 seconds

Overlay histogram showing SH-SY5Y cells stained with ab75524 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab75524, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ICC/IF image of ab75524 stained Hek293 cells. The cells were 4% PFA fixed (10mins) 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 (ab75524, 1:1000 dilution) overnight at +4°C. The secondary antibody (green) was ab96879 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.