- 701148 - Invitrogen Antibodies ADIPOQ antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Adiponectin Recombinant Rabbit Monoclonal Antibody (Product # 701148) and a 30 kDa band corresponding to Adiponectin was observed. Whole cell extracts (30 µg lysate) of (Fig. a) 3T3-L1 (Lane 1), 3T3-L1 treated with PTI (1X, 4hrs) (Lane 2), 3T3-L1 differentiated to Adipocytes (Lane 3), 3T3-L1 differentiated to Adipocytes further treated with PTI (1X, 4hrs) (Lane 4), ADSC (Lane 5), ADSC treated with PTI (1X, 4hrs) (Lane 6), ADSC differentiated to Adipocytes (Lane 7), ADSC differentiated to Adipocytes further treated with PTI (1X, 4hrs) (Lane 8), A549 (Lane 9), A549 treated with PTI (1X, 4hrs) (Lane 10); (Fig. b) Mouse Adipose (Lane 1), Rat Adipose (Lane 2), Mouse Brown Adipose (Lane 3), Rat Brown Adipose (Lane 4), Mouse Kidney (Lane 5), Rat Kidney (Lane 6), Mouse Liver (Lane 7), Rat Liver (Lane 8), Mouse Brain (Lane 9) Rat Brain (Lane 10) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were 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, 1:20000 dilution). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Adiponectin in whole cell extracts of serum-starved 3T3 L1 cells treated with insulin (100 ng/mL, 15 min) using an Adiponectin recombinant rabbit monoclonal antibody (Product # 701148) at a dilution of 1 µg/mL. Samples were detected using chemiluminescence (ECL). Results show a band at ~28 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Adiponectin in HeLa cells using an Adiponectin recombinant rabbit monoclonal antibody (Product # 701148) followed by detection using an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (green) (Image A). Nuclei were stained using DAPI (Image B) and actin stained with Alexa Fluor 594 phalloidin (red) (image C). Image D is a composite image showing sub cellular localization in the perinuclear region.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Adiponectin was performed using 70% confluent log phase ADSC cells differentiated to Adipocytes further treated with PTI (1X for 4hrs).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 Adiponectin Recombinant Rabbit Monoclonal Antibody (11H4L4) (Product # 701148) at 5 µg/mL 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, e, i, m: Green). Nuclei (Panel b, f, j, n :Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c, g, k, o: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel m represents the merged images showing cytoplasmic localization. Panel (a-d) represent ADSC cells ,Panel (e-h) represents ADSC cells treated with PTI (1X for 4hrs), Panel (i-l) represents ADSC cells differentiated to Adipocytes, Panel (m-p) represents ADSC cells differentiated to Adipocytes then further treated with PTI The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Adiponectin was performed using 70% confluent log phase ADSC differentiated to Adipocytes then treated with PTI (1X for 4hrs). 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 Adiponectin Recombinant Rabbit Monoclonal Antibody (11H4L4) (Product # 701148) at 5 µg/mL 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,e,i: Green) represents untreated cells ADSC,ADSC differentiated to Adipocytes and A549 cells, (Panel b, f, j) represents the merged images for the respective images for untreated cells, Similarly (Panel c, g ,k: Green) represents cells treated with PTI for ADSC, ADSC differentiated to Adipocytes and A549, (Panel d ,h, l ) represents the merged images for the respective images for PTI treated cells. Panel h represents the merged image showing cytoplasmic localization in ADSC adipocyte differentiated cells upon PTI treatment. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Adiponectin showing staining in the cytoplasm and membrane of paraffin-embedded human kidney tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Adiponectin Recombinant Rabbit Monoclonal Antibody (Product # 701148) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

701148

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