- 700489 - Invitrogen Antibodies CCL2 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of recombinant MCP-1 (100 ng/lane) using a MCP-1 recombinant rabbit monoclonal antibody (Product # 700489) at a dilution of 0.1 µg/mL. NBT/BCIP was used as the substrate (Product # WB7105).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-MCP-1 Recombinant Rabbit Monoclonal Antibody (29H86L56)(Product # 700489) and a 11 kDa band corresponding to MCP-1 was observed in THP-1 treated with PMA and LPS followed by PTI. Non specific bands was also observed in THP-1 around 50 kDa, 110 kDa and 150 kDa. Whole cell extracts (30 µg lysate) of THP-1 (Lane 1), THP-1 treated with PTI (1X for 4hr) (Lane 2), THP-1 treated with PMA (50 ng/mL for 16hr) and LPS (100 ng/mL for 1hr) followed by PTI (1X for 4hr) (Lane 3) were electrophoresed using Novex™ 16% Tricine Protein Gel (Product # EC6695BOX). 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 (0.3 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 SuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of CCL2/MCP-1 was performed using 70% confluent log phase HCT 116 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 CCL2/MCP-1 (29H86L56) Recombinant Rabbit Monoclonal Antibody (Product # 700489) 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 represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of MCP-1 was performed using 70% confluent log phase U-937 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 MCP-1 Recombinant Rabbit Monoclonal Antibody (29H86L56) (Product # 700489) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731), (1:2000), 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 cytoplasmic(Golgi complex like pattern) 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)
Main image
Experimental details: Flow cytometry analysis of CCL2 / MCP-1 was done on HCT 116 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 MCP-1 ABfinity™ Rabbit Monoclonal Antibody (700489, 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.

700489