LF-MA0201

antibody from Invitrogen Antibodies

Targeting: SHC1 p66, SHC, ShcA

ELISA

Immunocytochemistry

Immunoprecipitation

Antibody data

Submit

Validation data

SHC1 protein structure - LF-MA0201 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of SHC was achieved by transfecting A431 cells with SHC specific siRNAs (Silencer® select Product # s12812, s12813 and s28721). Western blot analysis (Fig a) was performed using membrane extracts from the SHC knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Anti-SHC Mouse Monoclonal Antibody (Product # LF-MA0201, 1 µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:5000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to SHC.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of COS-7 (Lane 1), MKN45 (Lane 2), Jurkat (Lane 3), Caco-2 (Lane 4), U-87 MG (Lane 5), KARPAS 299 (Lane 6) and NTERA-2 (Lane 7). The blot was probed with Anti-SHC Mouse Monoclonal Antibody (Product # LF-MA0201, 1 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). Two isoforms of 51 and 47 kDa corresponding to SHC were observed across the cell lines tested. In addition to this a 62 kDa isoform was observed in COS-7 and U-87 MG cell lines. 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® 2 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: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of A-431 (Lane 1), A549 (Lane 2), H1975 (Lane 3), MKN45 (Lane 4), Caco-2 (Lane 5), Jurkat (Lane 6), Hep G2 (Lane 7), COS-7 (Lane 8), NTERA-2 (Lane 9) and U-87 MG (Lane 10). The blot was probed with Mouse Anti- SHC Monoclonal antibody (Product # LF-MA0201, 1 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Bands at 47, 51 and 62 kDa corresponding to different isoforms of SHC were observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), 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® 2 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).