Western blot analysis was performed on whole cell extracts (30 µg lysate) of SH-SY5Y (Lane 1), IMR-32 (Lane 2), Rat Brain (Lane 3) and Mouse Brain (Lane4). The blots were probed with Anti- SCG10/Stathmin-2 Rabbit Polyclonal Antibody (Product # 720178, 0.5-1 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 21 kDa band corresponding to SCG10/Stathmin-2 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), 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).
Immunofluorescence was performed on fixed and permeabilized SHSY-5Y cells for detection of Stathmin-2 using Anti-Stathmin-2 Rabbit Polyclonal Antibody (Product # 720178, 1 µg/mL), alpha-Tubulin was detected using Anti-alpha Tubulin Monoclonal Antibody (Product # 32-2500, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (Heavy Chain) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000), Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor®594conjugate (Product # A-11032, 1:400) respectively. Panel a) shows representative cells that were stained for detection and localization of Stathmin-2 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938,). Panel c) represents cytoskeletal alpha-tubulin staining (red). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of Stathmin-2. Panel e) represents control cells with no primary Antibody to assess background.
Immunofluorescence was performed on fixed and permeabilized SHSY-5Y cells for detection of Stathmin-2 using Anti-Stathmin-2 Rabbit Polyclonal Antibody (Product # 720178, 1 µg/mL), alpha-Tubulin was detected using Anti-alpha Tubulin Monoclonal Antibody (Product # 32-2500, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000), Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor®594conjugate (Product # A-11032, 1:400) respectively. Panel a) shows representative cells that were stained for detection and localization of Stathmin-2 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938,). Panel c) represents cytoskeletal alpha-tubulin staining (red). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of Stathmin-2. Panel e) represents control cells with no primary Antibody to assess background.
Supplier Page from Thermo Fisher Scientific for STMN2 Antibody