- 701306 - Invitrogen Antibodies MMP16 antibody

Submitted references Exosomal miR-193a and let-7g accelerate cancer progression on primary colorectal cancer and paired peritoneal metastatic cancer.

Cho WC, Kim M, Park JW, Jeong SY, Ku JL
Translational oncology 2021 Feb;14(2):101000

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MMP16 was performed by loading 30 µg of Jurkat and MDA-MB-231 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. MMP16 was detected at ~55 kDa using MMP16 Recombinant Rabbit Monoclonal Antibody (Product # 701306) at a 1:500 dilution in 2.5% skim milk at 4°C overnight on a rocking platform. Detection was performed using an HRP-conjugated Goat anti-Rabbit secondary antibody (Product # G-21234) at a 1:5000 dilution and 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: Western blot was performed using Anti-MMP16 Recombinant Rabbit Monoclonal Antibody (13H7L7) (Product # 701306) and a 53kDa band corresponding to Matrix metalloproteinase-16 was observed across cell lines and tissue extracts tested except Mouse Lung, Rat Lung and Mouse Bain which is reported to be low. Fig.a shows membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), DU 145 (Lane 2), PC-3 (Lane 3), LNCaP (Lane 4), A549 (Lane 5), Jurkat (Lane 6), U-87 MG (Lane 7) and Fig.b shows tissue extracts (30 µg lysate) of Mouse Pup Lung (Lane 1), Rat Pup Lung (Lane 2), Mouse Lung (Lane 3), Rat Lung (Lane 4), Mouse Pup Brain (Lane 5) and Mouse Brain (Lane 6) 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 (1:1000 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 FL 1000 (Product # A32752). 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 MMP-16 in whole cell extracts from Jurkat cells (lane 1) and MDAMB-231 cells (lane 2) using a MMP-16 recombinant rabbit monoclonal antibody (Product # 701306) at a dilution of 2 µg/mL. Samples were detected using chemiluminescence (ECL). Results show a band at ~52kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of MMP16 was performed on 80% confluent log phase HeLa cells. 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 MMP16 Recombinant Rabbit Monoclonal Antibody (Product # 701306) at a dilution of 1:1000 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) and panel d is a merged image showing membrane, cytoplasmic and traces of nuclear localization. The images were captured using a Nikon microscope at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of MMP-16 showing staining in the cytoplasm of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM 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 MMP-16 monoclonal antibody (Product # 701306) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of MMP-16 showing staining in the Cytoplasm 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 10 mM 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 MMP-16 monoclonal antibody (Product # 701306) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of MMP-16 showing staining in the cytoplasm of paraffin-embedded rat kidney tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM 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 MMP-16 monoclonal antibody (Product # 701306) diluted in 3% BSA-PBS at a dilution of 1:200 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of MMP16 was performed on HeLa cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0. 25% Tritonª X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with ABfinityª MMP-16 recombinant rabbit monoclonal antibody (Product # 701306, red histogram) or with rabbit isotype control (pink histogram) at a dilution of 1:400 in 2.5% BSA. After incubation at room temperature for 3 hours, the cells were labeled with Alexa Fluor¨ 488 goat anti-Rabbit Secondary antibody (Product # 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 MMP-16 in HeLa cells using a MMP-16 recombinant rabbit monoclonal antibody (Product # 701306). 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 (right peak) compared to a control without primary antibody (left peak).

701306

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