- 701028 - Invitrogen Antibodies IL6 antibody

Huang K, Li W, Chen Y, Zhu J
Oncology letters 2018 Dec;16(6):7068-7073

Buchegger P, Preininger C
Analytical chemistry 2014 Mar 18;86(6):3174-80

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of recombinant human IL6 (1 ng/lane) using an IL-6 recombinant rabbit monoclonal antibody (Product # 701028) at a dilution of 0.1 µg/mL. NBT/BCIP was used as the substrate (Product # WB7105).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-IL-6 Recombinant Rabbit Monoclonal Antibody (4H16L21) (Product # 701028) and a 24 kDa band corresponding to IL-6 was observed across upon treatment of HUVEC cells with LPS and Brefeldin A. Whole cell extracts (30 µg lysate) of HUVEC (Lane 1) and HUVEC treated with LPS and Brefeldin A (0.5 µg/mL LPS for 24 h and 300 ng/mL Brefeldin A for last 20 h of LPS stimulation) (Lane 2) 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 µg/mL dilution) 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 Reagent Kit (Product # WP20005).Expression of IL-6 was observed to be to be upregulated upon treatment of HUVEC cells with LPS and Brefeldin A. An uncharacterized band (*) was observed at ~55 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on recombinant proteins, 20 ng of IL-6 (Lane 1) and 50 ng of IL-6 (Lane 2). The blots were probed with Rabbit Monoclonal Anti-IL6 Antibody (Product # 701028, 0.1-1 µg/mL) and detected by chemiluminescence Goat anti-Rabbit IgG Secondary Antibody, HRP conjugate (Product # G-21234, 1:5000 dilution). A 21 kDa band corresponding to IL6 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 by 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: Immunofluorescence analysis of IL-6 was performed using 70% confluent log phase HUVEC cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with IL-6 Recombinant Rabbit Monoclonal Antibody (4H16L21) (Product # 701028) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing predominantly Golgi Complex and Endoplasmic Reticulum localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of IL-6 on LPS treated THP-1 cells. Cells were fixed with 4% formaldehyde for 30 min on ince and permeabilized with IC permeabilization buffer (Product # PB001). After incubation with blocking buffer (Product # 37525) for 30 min on ice, cells were then stained with anti-IL-6 mouse monoclonal antibody (Product # 701028) or IgG control at 1:100 dilution with IC permeabilization buffer for 30 min on ice. After washing with ice-cold IC permeabilization buffer for 3 times, the cells were stained with DyLight 488 goat anti-mouse secondary antibody (Product # 35502) for 30 min on ice. A representative 10,000 cells were acquired for each sample.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of IL-6 on PHA-M stimulated THP-1 cells with or without LPS treatment. Cells were fixed with 4% formaldehyde for 30 min on ince and permeabilized with IC permeabilization buffer (Product # PB001). After incubation with blocking buffer (Product # 37525) for 30 min on ice, cells were then stained with anti-IL-6 rabbit monoclonal antibody (Product # 701028) at indicated dilution with IC permeabilization buffer for 30 min on ice. After washing with ice-cold IC permeabilization buffer for 3 times, the cells were stained with DyLight 488 goat anti-rabbit secondary antibody (Product # 35552) for 30 min on ice. A representative 10,000 cells were acquired for each sample.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of IL-6 on LPS treated THP-1 cells. Cells were fixed with 4% formaldehyde for 30 min on ince and permeabilized with IC permeabilization buffer (Product # PB001). After incubation with blocking buffer (Product # 37525) for 30 min on ice, cells were then stained with anti-IL-6 rabbit monoclonal antibody (Product # 701028) at indicated dilution with IC permeabilization buffer for 30 min on ice. After washing with ice-cold IC permeabilization buffer for 3 times, the cells were stained with DyLight 488 goat anti-rabbit secondary antibody (Product # 35552) for 30 min on ice. A representative 10,000 cells were acquired for each sample.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. ELISA. ELISA was used to detect the expression of IL-1 and IL-6 in two groups of cells. The OD values of IL-1 and IL-6 in HeLa cells treated with 10 ug/ml PM2.5 were significantly higher than those in NC group. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Indirect ELISA was performed using various dilutions of Anti-IL-6 Rabbit Monoclonal Antibody (Product # 701028) to detect IL-6 recombinant protein coated onto the plate. A non-linear regression analysis was performed (4 PL) and LOD and LOQ for the antibody was determined.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. Western blotting. Compared with HeLa cells in NC group, the relative expression levels of (A) IL-1 and (B) IL-6 proteins in HeLa cells treated with 10 ug/ml PM2.5 were significantly increased. (C) Western blots of IL-1 and IL-6 protein in the two groups of cells are shown. *P

701028