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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [3]
- Immunocytochemistry [1]
- Flow cytometry [1]
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- Product number
- 701684 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- ATG12 Recombinant Rabbit Monoclonal Antibody (20H24L24)
- Antibody type
- Monoclonal
- Antigen
- Synthetic peptide
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Antibody clone number
- 20H24L24
- Vial size
- 100 µg
- Concentration
- 0.5 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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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 (Lane 1), HT-29 (Lane 2), Panc-1 (Lane 3), HCT 116 (Lane 4), Mouse Colon (Lane 5) and Mouse Pancreas (Lane6). The blots were probed with Anti-ATG12 Recombinant Rabbit Monoclonal Antibody (Product # 701684, 1-2 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 17 kDa band corresponding to ATG12 was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12% Bis-Tris gel (Product # NP0342BOX), 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 Rabbit Monoclonal Antibody (20H24L24) (Product # 701684,1:300) 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 Rabbit Monoclonal Antibody (20H24L24) (Product # 701684) and a 55 kDa band corresponding to ATG12-ATG5 complex and a 17 kDa band corresponding to ATG12 was observed across all the cell lines and tissues 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), HCT 116 (Lane 5), Mouse Colon (Lane 6) and Rat Colon (Lane 7) 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 (1:300) 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 Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence was performed on methanol fixed HeLa cells for detection of ATG12 using ATG12 Recombinant Rabbit Polyclonal Antibody (Product # 701684, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Cytoskeleton was stained with alpha-Tubulin Monoclonal Antibody (Product # 32-2500, 1 µg/mL) followed by Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor® 594 conjugate (Product # A-11032, 1:400). Panel a) shows 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 alpha-Tubulin staining (red). 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.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Flow Cytometry analysis of ATG12 was performed on HEK-293 cells labeled with ABfinity™ Anti-ATG12 Recombinant Rabbit Monoclonal Antibody (Product# 701684, 5 ug/ 1M cells) or with rabbit isotype control at 0.5 ug/ml and detected with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, (Alexa Fluor® 488 conjugate, Product # A27034, 0.4 ug/ml, 1:2500) as represented by the red and pink histograms respectively. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control. A representative of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (4468770).
