- PA5-19701 - Invitrogen Antibodies SNAP25 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Mouse Spinal Cord Tissue Lysate using Product # PA5-19701, SNAP25 primary antibody at a dilution of 1 µg/mL (lane 1). Product # PA5-19701 staining of Rat Spinal Cord Tissue Lysate at a dilution of 1 µg/mL (lane 2). Blot treated with a secondary IR Dye680-conjugated Goat polyclonal anti-Rabbit antibody was used at a dilution of 1:10000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-SNAP25 Polyclonal Antibody (Product # PA5-19701) and a 25 kDa band corresponding to Synaptosomal-associated protein 25 was observed across the cell lines and tissues tested. Whole cell extracts (30 µg lysate) of PC-12 (Lane 1), PC-12 treated with NGF and KCl (NGF at 200 nM, KCl at 50 nM for 7 days) (Lane 2) and tissue extracts of Mouse Heart (Lane 3), Rat Heart (Lane 4), Mouse Brain (Lane 5), Rat Brain (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent staining of SH-SY5Y cells using Product # PA5-19701, anti-SNAP25 antibody. The cells were fixed with PFA (4%) for 10 minutes, permabilised with BSA (1%), normal goat serum (10%) and glycine (0.3 M) in 0.1% T-BST for 20 minutes and exposed to the primary antibody at a concentration of 5 µg/mL for 1 hour at room temp. The secondary antibody was a 448 fluorescence conjugated Goat anti-rabbit IgG (green) at a dilution of 1:1000. A WGA- 594 fluorescent conjugated stain was used to label plasma membranes (red) and the nuclei stain was DAPI (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent staining of SH-SY5Y cells using Product # PA5-19701, anti-SNAP25 antibody. The cells were fixed with PFA (4%) for 10 minutes, permabilised with BSA (1%), normal goat serum (10%) and glycine (0.3 M) in 0.1% T-BST for 20 minutes and exposed to the primary antibody at a concentration of 5 µg/mL for 1 hour at room temp. The secondary antibody was a 448 fluorescence conjugated Goat anti-rabbit IgG (green) at a dilution of 1:1000. A WGA- 594 fluorescent conjugated stain was used to label plasma membranes (red) and the nuclei stain was DAPI (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Synaptosomal-associated protein 25 was performed using 70% confluent log phase PC-12 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with SNAP25 Polyclonal Antibody (Product # PA5-19701) at 0.8 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing enhanced cytoplasmic and membrane localization of SNAP-25 in PC-12 cells differentiated to neuronal phenotype with NGF (200 ng/mL 5 days). Panel e represents the undifferentiated PC-12 cells. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

PA5-19701