All lanes : Anti-SNAP23 antibody (ab3340) at 1 µg/ml

Lane 1 : Untransfected U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysates
Lane 2 : Non-specific scrambled siRNA transfected U-87 MG whole cell lysates
Lane 3 : SNAP23 KO U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysates

Secondary
All lanes : Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate at 0.25 µg/ml

Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to SNAP23.

Immunofluorescence analysis of SNAP-23 was performed using 90% confluent log phase U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) cells with ab3340. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ab3340 at 2µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with DAPI. F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membrane localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

ab3340 (1ug/ml) staining SNAP23 in human tonsil using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of cellular membrane compartments of the lymphatic nodules.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

All lanes : Anti-SNAP23 antibody (ab3340) at 2 µg/ml

Lane 1 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate
Lane 2 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate

Lysates/proteins at 30 µg/ml per lane.

Secondary
All lanes : Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate at 0.4 µg/ml

Anti-SNAP23 antibody (ab3340) + Rat brain tissue

ICC/IF image of ab3340 stained PC-12 (Rat adrenal gland pheochromocytoma cell line) cells. The cells were 4% PFA 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 (ab3340, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit 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.

Anti-SNAP23 antibody (ab3340) at 1 µg/ml + Recombinant Human SNAP23 protein (ab79180) at 0.001 µg

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

Developed using the ECL technique.

Performed under reducing conditions.

Exposure time: 2 minutes