- 720056 - Invitrogen Antibodies ATG3 antibody

No submitted references: Submit reference

No comments: Submit comment

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of ATG3 was achieved by transfecting HeLa with ATG3 specific siRNAs (Silencer® select Product # s34733, s34731). Western blot analysis (Fig. a) was performed using membrane enriched extracts from the ATG3 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with ATG3 Polyclonal Antibody (Product # 720056, 0.5µg/ml) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). 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 ATG3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of K-562 (Lane 1), THP-1 (Lane 2), A-549 (Lane 3), Jurkat (Lane4), Mouse Muscle (Lane 5) and Mouse Kidney (Lane 6). The blots were probed with Anti-ATG-3 Rabbit Polyclonal Antibody (Product # 720056, 0.5-1 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 36kDa band corresponding to ATG-3 was observed across cell lines and tissue 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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence was performed on fixed and permeabilized HeLa cells for detection of ATG-3 using Anti- ATG-3 Rabbit Polyclonal Antibody (Product # 720056, 1 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 ATG-3 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 Membrane and Cytoplasmic localization of ATG-3. 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 Atg-3 was performed on HeLa cells labeled with Anti-Atg-3 Rabbit Polyclonal Antibody (Product# 720056, 2-4 ug/ 1M cells) or with Rabbit isotype control and detected with Goat anti-Rabbit IgG (H+L) Superclonalª 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).

720056