Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [3]
- Immunocytochemistry [1]
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Validation data
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- Product number
- 710716 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- ATG12 Recombinant Polyclonal Antibody (20 HCLC)
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- This antibody is predicted to react with Monkey, Pig and Horse.
- Antibody clone number
- 20 HCLC
- Concentration
- 0.5 mg/mL
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on whole cell extracts (30 µg lysate) of HEK 293 (Lane1), THP-1 (Lane 2), PANC-1 (Lane 3), HCT-116 (Lane 4), C2C12 (Lane 5), NIH 3T3 (Lane 6), Mouse colon (Lane 7) and Mouse Kidney (Lane 8). The blots were probed with Anti-ATG12 Recombinant Rabbit Polyclonal Antibody (Product # 710716, 4-5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 55 kDa band corresponding to ATG12 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).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of ATG12 was achieved by transfecting U-2 OS with ATG12 specific siRNAs (Silencer® select Product # S17464, S17465). Western Blot analysis (Fig. a) was performed using Whole cell extracts from the ATG12 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The Blot was probed with ATG12 Recombinant Polyclonal Antibody (20 HCLC) (Product # 710716, 1:350 ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000). Densitometric analysis of this western Blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to ATG12.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot was performed using Anti-ATG12 Recombinant Polyclonal Antibody (20 HCLC) (Product # 710716) and a 55 kDa band corresponding to ATG12-ATG5 complex was observed across all the cell lines tested. Whole cell extracts (30 µg lysate) of U-2 OS (Lane 1), SH-SY5Y (Lane 2), Hep G2 (Lane 3), NIH/3T3 (Lane 4) and HCT 116 (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2002) by iBlot® 2 Dry Blotting System (Product # IB21001). The Blot was probed with the primary antibody (1:350) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence was performed on fixed and permeabilized HeLa for detection of ATG12 using Anti-ATG12 Recombinant Rabbit Polyclonal Antibody (Product # 710716, 2 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 ATG12 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of ATG12 Panel e) represents control cells with no primary Antibody to assess background.