Cells were cultured in complete DMEM on coverslips for 3 weeks after which the cell supernatant was pooled (n = 3) and stored at − 80 °C until further use. The experiment was carried out as outlined in the manufacturer’s instructions (Abcam Human MMP Antibody Array), where 1 mL of undiluted pooled sample was added to the membrane and incubated overnight. The protein of interest was captured by antibody array chips (“spots”), biotin-conjugated antibodies and then labelled streptavidin for detection. The membranes were analysed with an Azure c500 Infrared Western Blot Imaging System and analysed using Fiji software (ImageJ derivative, free download from NIH). Pixel density was calculated for each spot and then averaged.
Human mmp antibody array
Manufactured by Abcam
Sourced in United Kingdom
The Human MMP Antibody Array is a multiplex assay that allows for the simultaneous detection and quantification of multiple human matrix metalloproteinase (MMP) proteins in a single experiment. The array contains antibodies specific to various MMP targets, enabling researchers to profile the expression levels of these proteins in their samples.
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Lab products found in correlation
Chemidoc xrs system, by Bio-Rad (2 mentions)
Bca protein assay, by Bio-Rad (1 mentions)
Halt protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Quantity one analysis software, by Bio-Rad (1 mentions)
Proteome profiler array, by R&D Systems (1 mentions)
Fusion fx7 imager, by Vilber (1 mentions)
Luminata crescendo western hrp substrate, by Merck Group (1 mentions)
Mouse xl cytokine array kit, by R&D Systems (1 mentions)
Image studio lite, by LI COR (1 mentions)
Proteome profiler human xl cytokine array kit, by R&D Systems (1 mentions)
Quantikine elisa kit, by R&D Systems (1 mentions)
8 protocols using human mmp antibody array
1
Quantifying Secreted Proteases via Antibody Array
2
Quantitative Multiplex Analysis of MMPs and TIMPs
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The pre-enriched EVs and cell pellets were diluted in PBS, 1X RIPA lysis buffer. HaltTM Protease & Phosphatase Inhibitor cocktail (Thermo Scientific®, Waltham, MA, USA) were added, and the samples were incubated at 4 °C for 15 min. Protein was quantified using the BCA protein assay (BioRad®, Hercules, CA, USA). Human MMP Antibody Array (ab134004, Abcam®, Cambridge, UK) was used to measure MMPs and TIMPs. This ELISA-like multiplex approach offers substantial benefits for examining a defined set of proteins in parallel and detects seven human MMPs (MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10 and MMP-13) and three TIMPs (MMP-13, TIMP-1, TIMP-2 and TIMP-4). Activity was determined in EVs lysates using 100 ng of protein input. Quantification was performed by ChemiDocTM XRS+ System and data analysis was performed using Quantity One® Analysis Software (BioRad®, Hercules, CA, USA).
3
Quantitative Protease Profiling of Cell Samples
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Relative quantitative measurement of 40 unique proteases in cell lysates and cell culture media was determined using commercially available antibody arrays (Human MMP antibody array, ab197453 Abcam, Cambridge, UK; and Proteome ProfilerTM Array, ARY021 R&D Systems, Minneapolis, MN, USA) as per the manufacturer’s instructions. Each array was probed using 50 µg of total protein from either cell lysate or concentrated cell culture supernatant. Arrays were visualized using either a Typhoon Variable Mode Imager (Molecular Dynamics, Sunnyvale, CA, USA) equipped with a Cy3 wavelength filter (Human MMP array) or a ChemiDoc™ XRS+ System (BioRad, Hercules, CA, USA) to detect chemiluminescence (Proteome Profiler array). Signal intensities were determined using Fiji software v1.0 [54 (link),55 (link)], and replicate values were individually normalized against a reference array.
4
Exosomal MMP and TIMP Quantification
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Exosomal MMP and TIMP levels were measured using the Human MMP Antibody Array (Abcam), targetting 10 proteins, according to the manufacturer’s instructions. Exosome samples, previously isolated from 1 mL of conditioned media, were first lysed with RIPA 1X (Abcam) and resuspended in a total of 1 mL of PBS. Briefly, membranes were first blocked using the given Blocking Buffer and then incubated with the resuspended samples overnight at 4 °C. The following day, membranes were washed and incubated with the biotin-conjugated antibodies for 2 h at room temperature and then incubated in HRP-Conjugated Streptavidin overnight at 4 °C. Chemiluminescence was imaged using Fusion Fx7 imager (Vilber Lourmat), protein dots were quantified using ImageJ spot recognition analysis.
5
Quantifying Matrix Metalloproteinases in HASM Cells
6
MMP Antibody Array for Quantifying Secreted Proteases
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The human MMP antibody array (Abcam) was used according to the manufacturer’s instructions. Briefly, array membranes were incubated in equal quantities of the culture supernatant from PBS- or NS1-treated THP-1 cells or NS1-treated leukocytes for 24 h overnight at 4°C. After washing with commercial wash buffer, the membranes were incubated with biotin-conjugated anti-MMP antibodies, followed by HRP-conjugated streptavidin. Bound HRP-conjugated antibodies were detected using the Luminata Crescendo Western HRP substrate (Merck Millipore, Darmstadt, Germany).
7
Quantifying Matrix Metalloproteinases in HASM Cells
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Serum-starved HASM cells were treated with vehicle or Af (2 µg ml−1) for 24 hours. Relative amounts of MMPs and TIMPs in cell supernatants were determined using a Human MMP Antibody Array (Abcam,) according to the manufacturer’s instructions. Blots were developed using biotinylated primary antibodies and streptavidin and quantified using Image Studio software.
8
Cytokine and MMP Profiling of Brain Microvessels and Endothelial Cells
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The Mouse Cytokine Array XL Kit or Proteome Profiler Human XL Cytokine Array Kit (R&D Systems, Minneapolis, USA) was used to analyze different cytokines, interleukins, chemokines, and acute-phase proteins in mouse brain microvessels or hCMEC/D3 cell lysates stimulated with 20% human serum. The Human MMP Antibody array (Abcam) was used to analyze MMPs and TIMPs in human serum or hCMEC/D3 cell lysates stimulated with 20% human serum. These kits are membrane-based sandwich immunoassays. Briefly, membranes were blocked with blocked buffer for 1 h followed by overnight incubation with samples at 2-8°C. Membranes were incubated with a detection antibody cocktail. Bound antibodies were detected using Streptavidin-HRP and Chemi Reagent mix. Image Studio™ Lite (LI-COR Biosciences, USA) software was used to analyze the data. Mouse serum TIMP-1 levels were measured using a Quantikine ELISA kit (R&D Systems). Human serum IL-6 and TNF-α were measured using a multiplex Human Luminex Assay (R&D Systems) on the Luminex MAGPIX System (Luminex Corporation).
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