ab41455 staining SNAP25 in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab41455 at 1µg/ml and ab192757, Mouse mono Anti-PSD95 antibody [K28/43] - Synaptic Marker. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

All lanes : Anti-SNAP25 antibody (ab41455) at 1 µg/ml

Lane 1 : Spinal cord (Rat) tissue lysate
Lane 2 : Hippocampus (Mouse) tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 23 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?

Anti-SNAP25 antibody (ab41455) at 1 µg/ml + Recombinant Human SNAP25 protein (ab74529) at 0.01 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 23 kDa


Exposure time: 1 minute

ab4455 staining SNAP25 in PC12 cells. The cells were fixed with 100% methanol (5 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 ab41455 at 5µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.

All lanes : Anti-SNAP25 antibody (ab41455) at 1 µg/ml

Lane 1 : Marker
Lane 2 : Zebrafish brain homogenate at 20 µg
Lane 3 : SH-SY5Y (Human neuroblastoma cell line) whole cell lysate at 20 µg
Lane 4 : Mouse brain homogenate at 20 µg

Secondary
All lanes : Goat polyclonal to Rabbit IgG – H&L – Pre-Adsorbed (HRP) at 1/6000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 23 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?


Exposure time: 2 minutes

SNAP25 was immunoprecipitated using 0.5mg Rat Spinal Cord tissue lysate, 5µg of Rabbit polyclonal to SNAP25 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, Rat Spinal Cord 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 ab41455.
Secondary: Clean-Blot IP Detection Reagent (HRP) at 1/500 dilution.
Band: 26kDa; SNAP25