- 710915 - Invitrogen Antibodies ATG4B antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of ATG4B was performed by loading 30 µg of HeLa (lane 1), HEK 293 (lane 2), Jurkat (lane 3), HCT 116 (lane 4), COS 7 (lane 5), MDCK (lane 6) and HepG2 (lane 7) cell lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800) and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature on a rocking platform. ATG4B was detected at ~44 kDa using ATG 4B Recombinant Rabbit Polyclonal Antibody (Product # 710915) at 1-2 µg/mL in 5 % skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG - HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and 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 of ATG4B was performed by loading 30 µg of HeLa (lane 1), HEK 293 (lane 2), Jurkat (lane 3), HCT 116 (lane 4), COS 7 (lane 5), MDCK (lane 6) and HepG2 (lane 7) cell lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800) and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature on a rocking platform. ATG4B was detected at ~44 kDa using ATG 4B Recombinant Rabbit Polyclonal Antibody (Product # 710915) at 1-2 µg/mL in 5 % skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG - HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and 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 of ATG4B was performed by loading 30 µg of HeLa (lane 1), HEK 293 (lane 2), Jurkat (lane 3), HCT 116 (lane 4), COS 7 (lane 5), MDCK (lane 6) and HepG2 (lane 7) cell lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800) and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature on a rocking platform. ATG4B was detected at ~44 kDa using ATG 4B Recombinant Rabbit Polyclonal Antibody (Product # 710915) at 1-2 µg/mL in 5 % skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG - HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of ATG4B was achieved by transfecting MCF7 with ATG4B specific siRNAs (Silencer® select Product # s23244, s23246). Western blot analysis (Fig. a) was performed using whole cell extracts from the ATG4B knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with ATG4B Recombinant Polyclonal Antibody (Product # 710915, 1 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 0.25µg/ml, 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 ATG4B.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-ATG4B Recombinant Polyclonal Antibody (1HCLC) (Product # 710915) and a 42 kDa band corresponding to ATG4B was observed across all the cell lines tested. Whole cell extracts (30 µg lysate) of Jurkat (Lane 1), PC-3 (Lane 2), MCF7 (Lane 3), NIH/3T3 (Lane 4), SK-BR-3 (Lane 5), MDA-MB-231 (Lane 6) and Daudi (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) 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: Immunofluorescent analysis of ATG4B was done on 70% confluent log phase Rapamycin treated HeLa cells (treated with 100nM of Rapamycin A for 4 hours). The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with ATG14 Recombinant Rabbit Polyclonal Antibody (Product # 710915) at 2 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing nuclear localization. Panel e shows untreated HeLa cells. Panel f shows no primary antibody control. The images were captured at 20X magnification.

710915