All lanes : Anti-Synaptotagmin antibody [ASV30] (ab13259) at 1/1000 dilution

Lane 1 : Molecular weight ladder
Lane 2 : Lysates prepared from mouse brain
Lane 3 : Lysates prepared from rat brain

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ICC/IF image of ab13259 stained PC12 cells. The cells were 4% formaldehyde fixed (10 min) 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 (ab13259, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 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.

This image was generated using the ascites version of the product.

Synaptotagmin was immunoprecipitated using 0.5mg Mouse Brain tissue lysate, 5µg of Mouse monoclonal to Synaptotagmin and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Mouse Brain tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab13259.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.
Band: 64kDa; Synaptotagmin

This image was generated using the ascites version of the product.