- 700113 - Invitrogen Antibodies MAP3K7 antibody

Submitted references Analysis of copy number alterations reveals the lncRNA ALAL-1 as a regulator of lung cancer immune evasion.

Athie A, Marchese FP, González J, Lozano T, Raimondi I, Juvvuna PK, Abad A, Marin-Bejar O, Serizay J, Martínez D, Ajona D, Pajares MJ, Sandoval J, Montuenga LM, Kanduri C, Lasarte JJ, Huarte M
The Journal of cell biology 2020 Sep 7;219(9)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Mitogen-activated protein kinase kinase kinase 7 was achieved by transfecting HEK-293 with Mitogen-activated protein kinase kinase kinase 7 specific siRNAs (Silencer® select Product # S13768, S13766). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Mitogen-activated protein kinase kinase kinase 7 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with TAK1 Recombinant Rabbit Monoclonal Antibody (28H25L68) (Product # 700113, 1:2000 dilution ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20000 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 Mitogen-activated protein kinase kinase kinase 7.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-TAK1 Recombinant Rabbit Monoclonal Antibody (28H25L68) (Product # 700113) and a ~65 kDa band corresponding to Mitogen-activated protein kinase kinase kinase 7 was observed along with an uncharacterised band (*) at ~40 kDa across cell lines and tissues tested. Whole cell extracts (30 µg lysate) of A-431 (Lane 1), HeLa (Lane 2), K-562 (Lane 3), HEK-293 (Lane 4), HEK-293 treated with IL1-alpha (Lane 5), NIH/3T3 (Lane 6), PC-12 (Lane 7) and Mouse Testis (Lane 8) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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 (1:2000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:20000 dilution using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HeLa (Lane 1), CaCo-2 (Lane 2), HEK-MSR (Lane 3), A-431 (lane 4) and A549 (lane 5). The blots were probed with Recombinant Rabbit Monoclonal Anti-TAK1 Antibody (Product # 700113, 0.5-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Recombinant Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 67 kDa band corresponding to TAK1 was observed across cell lines tested along with an extra band at ~ 30 kDa. 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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of TAK1 in HeLa cells using a TAK1 recombinant rabbit monoclonal antibody (Product # 700113) at a dilution of 1 µg/mL followed by detection using an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody at a dilution of 1:1000 (green) and nuclei stained using Hoescht (blue). Pre-incubation with immunogen decreased signal (right).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of TAK1/MAP3K7 was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with TAK1/MAP3K7 (28H25L68), Recombinant Rabbit Monoclonal Antibody (Product # 700113) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 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® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of TAK1 was done on HEK cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with ABfinity™ TAK1 Recombinant Rabbit Monoclonal Antibody (700113, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of TAK1 in Jurkat cells using a TAK1 recombinant rabbit monoclonal antibody (Product # 700113) at a dilution of 2 µg. Cells were fixed and permeabilized using FIX & PERM (Product # GAS-004) reagent, and detection was performed using an Alexa Fluor 488 goat anti-rabbit IgG (black) compared to a control without primary antibody (gray) and incubated with the immunogenic protein (red).

700113

Antibody data