Human recombinant full-length OPN (flOPN) (Peprotech, Rocky Hill, NJ) was used for cell experiments. To obtain MMP-cleaved OPN (MMP-cOPN), 1 mg of OPN per ml was incubated with 0.5 ng per ml of MMP-7 (Millipore, Billerica, MA) for 4 h at 37 C in assay buffer (50 mM Tris, 150 mM NaCl, CaCl 2 2.5 mM, Brij-35 0.05%, pH adjusted to 7.6 with HCl) as earlier described (10) , of note the related "vehicle" controls included the same amount of MMP-7. Cleavage was evaluated qualitatively by immunoblotting according to Ref. 10) using a polyclonal anti-OPN antibody (RD-Systems, Minneapolis, MN).
Mmp 7
Manufactured by Merck Group
Sourced in United States, Morocco
MMP-7 is a laboratory reagent used for the detection and quantification of Matrix Metalloproteinase-7 (MMP-7) in biological samples. MMP-7 is an enzyme involved in the breakdown of extracellular matrix and is associated with various physiological and pathological processes. The MMP-7 product provides researchers with a tool to measure MMP-7 levels for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Mmp 9, by Merck Group (2 mentions)
Anti opn antibody, by R&D Systems (1 mentions)
Silver staining kit, by Thermo Fisher Scientific (1 mentions)
Recombinant human mmp 2, by Merck Group (1 mentions)
Gm6001, by Bio-Techne (1 mentions)
Human cellular fibronectin, by Merck Group (1 mentions)
Aurora a, by Abcam (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Chemiluminescence kit, by Santa Cruz Biotechnology (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
E cadherin, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibody, by Santa Cruz Biotechnology (1 mentions)
9 protocols using mmp 7
1
Generation and Validation of MMP-Cleaved OPN
2
Shielding sFlt from MMP-7 Degradation
3
In vitro Protein Digestion Assay
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Unless otherwise noted, in vitro digests were performed as previously described using 2.5 μg recombinant N-cdh and 0.5 μg recombinant active enzyme [5 (link)]. Recombinant human MMP-2 and MMP-7 were purchased from EMD Millipore (catalog numbers PF023 and 444270, respectively; Billerica, MA), while recombinant human ADAM-10 was purchased from R & D Systems (936-AD; Minneapolis, MN). The MMP activity inhibitor GM-6001 was purchase from Tocris (catalog number 2983; Bristol, U.K.) and used at a final concentration of 2 μM. Reactions were terminated by addition of denaturing loading buffer and a 95 °C incubation (5 min) followed by freezing. Digestion products were resolved by electrophoresis on a 4–15% Tris-glycine polyacrylamide gradient gel and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was subsequently stained with Coomassie Blue.
4
Fibronectin Cleavage by MMPs
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Plasma bovine fibronectin (5 μg, Sigma), human cellular fibronectin (5 μg, Sigma) and rat and human fibronectin aggregates (5 μg, see above) were respectively incubated with 100 ng recombinant human active MMP3 (Abcam, Cambridge, UK), MMP7 (Millipore, Darmstadt, Germany), and MMP9 (Millipore, San Diego, CA) in 50 µl MMP‐reaction buffer (50 mM Tris‐HCl, 0.15 M NaCl, 5 mM CaCl2, 0.05% Brij‐35, pH 7.5) at 37°C for 24 hr. For Western blot analysis, the reaction was terminated by adding non‐reducing SDS sample buffer. For coating purposes, the enzymatic activity of MMP7 was terminated by heating at 95°C for 10 min. Alternatively, fibronectin structural variants were incubated with PBS or cell conditioned media (35 µl) in the presence or absence of general MMP‐activator, 4‐aminophenylmercuric acetate (APMA, 2 mM, Sigma) in 50 μl MMP reaction buffer and incubated at 37°C for 72 hr.
5
Tissue Protein Extraction and Immunoblotting
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For tissue protein extraction, frozen samples were homogenized in RIPA lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, and 0.1% SDS). The protein concentration in each sample was estimated by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Immunoblotting was performed according to standard procedures. Antibodies used in this study include Aurora-A (monoclonal; Epitomics, Burlingame, CA, USA), MMP-7 (monoclonal; Millipore), MMP-10 (monoclonal; Millipore), FLJ10540 (generated by us) and β-actin (monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The first antibodies were detected by incubation with secondary antibodies conjugated to HRP (Bio/Can Scientific, Mississauga, ON, Canada) and developed using Western Lighting Reagent. The proteins were explored by X-ray films. The protein expression levels were quantified by ImageJ Software and represented as the densitometric ratio of the targeted protein to GAPDH or β-actin.
6
Western Blot Analysis of E-cadherin and MMP-7
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For the Western blot analysis, we mixed 10 ug of protein from each sample with 5 × Laemmli buffer and 5% β-mercaptoethanol and boiled it for 10 min. Briefly, we separated an equal volume of protein by SDS–polyacrylamide gel electrophoresis and transferred it to a nitrocellulose membrane. We then incubated the membranes overnight at 4 °C with a 1:1000 dilution of E-cadherin (clone number H-108, catalog number sc-7870; Santa Cruz Biotechnology, Inc, Dallas, Tx, USA), MMP-7 (catalog number AV46075; Sigma-Aldrich, St. Louis, MO, USA), or a 1:2000 dilution of β-actin antibody (clone number C4, catalog number sc-47778, Santa Cruz Biotechnology) for loading control in a blocking solution. Next, we incubated the membranes with a 1:1000 dilution of the appropriate anti-rabbit (catalog number sc-2004; Santa Cruz Biotechnology) or anti-mouse antibody (catalog number sc-2005; Santa Cruz Biotechnology) in the blocking solution. We viewed blots using the chemiluminescence kit (Santa Cruz Biotechnology), and captured the images with ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA).
7
Protein Expression Analysis by Western Blot
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For western blot, 60ug protein was added to SDS-PAGE, which was transferred to PVDF membranes (Millipore, MA, USA). Then, the membranes were incubated with the following primary antibodies at 4°C overnight: Eya2 (1:1000; Sigma, USA), MMP7, Bxl-2, AKT, p-AKT, caspase3, cleaved-caspase3, PARP and cleaved-PARP (1:1000; Cell Signaling Technology, USA), and GAPDH (1:2000; Cell Signaling Technology, USA). This was followed by secondary antibody incubation with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000, Santa Cruz), and imaging on the imaging system (DNR, Isereal).
8
Quantitative Analysis of MMPs Expression
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Total RNA was extracted from cells by using a TRIzol kit; then 2 μg of RNA was used to synthesize complementary DNA (cDNA) by using reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative real-time polymerase chain reaction (qPCR) was conducted using SYBR Green (KAPA Biosystems, Woburn, MA, USA) according to the manufacturer protocol, and reactions were performed using a StepOnePlus machine (Applied Biosystems, Foster City, CA, USA). The reaction conditions were 10 min at 95°C for polymerase activation and 40 cycles of 15 s at 95°C and 60 s at 60°C. Human MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-12, MMP-13, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), purchased from Sigma-Aldrich (St. Louis, MO, USA), were used as primers to amplify target genes. The expression levels of MMPs were determined by normalizing them to that of GAPDH. The threshold cycle (Ct) was set above the nontemplate control background and within the linear phase of target gene amplification to calculate the cycle numbers at which the transcript was detected (denoted Ct). Each sample was assayed in triplicate and the data displayed represent 3 independent experiments.
9
Protein Expression Analysis in Mammary Cells
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Mammary cells HBL-100 and BC BT474/MCF-7 cells were added to the pre-chilled cell lysate and centrifuged at 14,000 rpm for 10 min at 4°C, and the supernatant was aspirated to determine the protein content. Briefly, the extracted proteins were separated by 10% polyacrylamide gels and transferred to polyvinylidenefluoride membranes. After being blocked with 5% Skim milk/PBST, the membrane was treated with primary antibodies including SHIP2 (1:1000, CST, USA, #2730), GSK-3β (1:1000, CST, USA, #9315), β-catenin (1:1000, CST, USA, #8480), LEF (1:1000, CST, USA, #2230), Bcl-2 (1:1000, Santa Cruz Biotechnology, USA, #sc-509), Bax (1:1000, Santa Cruz Biotechnology, USA, #sc-20067), Caspase-3 (1:1000, Santa Cruz Biotechnology, USA, #sc-271759), cyclin D1 (1:1000, Santa Cruz Biotechnology, USA, #sc-8396), MMP-7 (1:1000, Sigma Aldrich, USA, #SAB1406133), AXIN2 (1:1000, Sigma Aldrich, USA, #SAB1100676) and CD44 (1:1000, Sigma Aldrich, USA, #SAB1405590) at 4°C overnight. Then, the membrane was treated with secondary antibody including anti-rabbit IgG in goat (1:10,000, CST, USA, #14708) at room temperature for 1 hr. After washing the film, ECL luminescent reagent was added for exposure and color development. GAPDH (#5174, American CST) was used as an internal reference, and the results were expressed as the expression ratio of target protein to internal reference protein.
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