701991
antibody from Invitrogen Antibodies
Targeting: SNAP25
bA416N4.2, dJ1068F16.2, RIC-4, RIC4, SEC9, SNAP, SNAP-25
Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [3]
- Immunocytochemistry [2]
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Validation data
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- Product number
- 701991 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- SNAP25 Recombinant Rabbit Monoclonal Antibody (1H7L4)
- Antibody type
- Monoclonal
- Antigen
- Synthetic peptide
- Description
- This antibody is predicted to react with Monkey, Horse, Pig, Rabbit
- Antibody clone number
- 1H7L4
- Concentration
- 0.5 mg/mL
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of SNAP25 was achieved by transfecting PC12 cells with SNAP25 specific siRNA (Silencer® select Product # s13189 and s13190 ). Western blot analysis was performed using Whole cell extract from the SNAP25 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-SNAP25 Recombinant Rabbit Monoclonal Antibody (Product # 701991, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution).. Loss of signal upon siRNA mediated knock down confirms that antibody is specific to SNAP25.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on 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 (30 µg lysate) of Mouse Brain (Lane 3) and Rat Brain (Lane 4). The blots were probed with Anti-SNAP25 Recombinant Rabbit Monoclonal Antibody (Product # 701991, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:4000 dilution). A 25 kDa band corresponding to SNAP25 was observed across the cell lines and tissues tested. Along with the desired bands, non-specific band at 40 kDa was observed in the tissues tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®10% Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on 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 (30 µg lysate) of Mouse Brain (Lane 3) and Rat Brain (Lane 4). The blots were probed with Anti-SNAP25 Recombinant Rabbit Monoclonal Antibody (Product # 701991, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:4000 dilution). A 25 kDa band corresponding to SNAP25 was observed across the cell lines and tissues tested. Along with the desired bands, non-specific band at 40 kDa was observed in the tissues tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®10% Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- For immunofluorescence analysis, Mouse Hippocampal neurons were fixed and permeabilized for detection of endogenous SNAP25 using Anti-SNAP25 Recombinant Rabbit Monoclonal Antibody (Product # 701991, 5 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of SNAP25 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) is a composite image of Panels a and b clearly demonstrating localisation of SNAP25 in cell body and neuronal processes. The images were captured at 40X magnification.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- For immunofluorescence analysis, PC12 cells were fixed and permeabilized for detection of endogenous SNAP25 using Anti-SNAP25 Recombinant Rabbit Monoclonal Antibody (Product # 701991, 5 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) is stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938) and cytoskeletal F-actin (red) staining using Rhodamine Phalloidin (Product # R415, 1:300) Panel a-d) shows representative un-treated cells that were stained for detection and localization of SNAP25 protein (green) with reduced signal compared to panel e-h) clearly demonstrating enhanced cytoplasmic and membrane localisation of SNAP-25 in PC12 cells differentiated to neuronal phenotype with NGF (200 ng/mL 5 days). The images were captured at 60X magnification.