Total protein was extracted from the cells in RIPA lysis buffer as described previously [24 (link)]. Equal amounts of cell lysates (40 μg protein) were separated by 10% SDS/PAGE gel, then transferred on to PVDF membranes. Subsequently, the membranes were incubated with primary antibodies including anti-MMP-9 antibody (1:500, Santa Cruz Biotechnology), anti-TIMP1 antibody (1:500, Santa Cruz Biotechnology), and anti-β-actin antibody (1:1000, Sigma–Aldrich) overnight at 4 °C. Then, the membranes were incubated with secondary antibody conjugated to horseradish peroxidase (1:2000; Boster, Wuhan, China) for 1 h at room temperature. The blots were analyzed by chemiluminescence detection (ECL, Amersham).
Anti mmp 9 antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-MMP-9 antibody is a laboratory reagent used for the detection and quantification of the Matrix Metalloproteinase-9 (MMP-9) protein. MMP-9 is an enzyme involved in the breakdown of extracellular matrix and is implicated in various physiological and pathological processes. The antibody can be used in various immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry, to measure the levels of MMP-9 in biological samples.
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Lab products found in correlation
Anti mmp 2 antibody, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Secondary antibody conjugated to horseradish peroxidase, by Boster Bio (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Anti timp1 antibody, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Ab52237, by Abcam (1 mentions)
Ab61224, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Anti e cadherin antibody, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab92547, by Abcam (1 mentions)
Ab16502, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab13556, by Abcam (1 mentions)
Ab86299, by Abcam (1 mentions)
Quantity one v4, by Bio-Rad (1 mentions)
Goat anti rabbit igg antibody conjugated with horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
8 protocols using anti mmp 9 antibody
1
Western Blot Analysis of MMP-9 and TIMP1
2
Western Blot Analysis of Protein Expression
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Total proteins were lysed in radioimmunoprecipitation (RIPA) buffer (Beyotime Institute of Biotechnology, Haimen, China). The concentrations of proteins were detected using the Bicinchoninic Acid (BCA) protein assay kit (Thermo Fisher Scientific, Inc.). Equivalent proteins (40 μg) were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck KGaA, Darmstadt, Germany). Membranes were incubated with primary antibodies, and secondary antibody (cat no LK2003L, Sungene Biotech Co., Ltd, China). The protein was detected using the enhanced chemiluminescence (ECL) substrate kit (Thermo Scientific Pierce) and ImageJ software (NIH, Bethesda, MD, USA). The results were analyzed by densitometry using Quantity One V4.6.2 software (Bio-Rad, USA). The primary antibodies were anti-TLR4 antibody (1: 1000, Abcam, ab13556); anti-COX-2 antibody (1: 1000, Abcam, ab52237); anti-TIMP-1 antibody (1: 1000, Abcam, ab61224); anti-MMP-2 antibody (1: 1000, cat. no. PF023, Millipore, Darmstadt, Germany); anti-MMP-9 antibody (1: 50, Santa Cruz, cat no. sc-21733); anti-E-cadherin antibody (1: 500, Abcam, ab76055); anti-vimentin antibody (1: 800, Abcam, ab92547); anti-p-p65 antibody (1: 1000, Abcam, ab86299); anti-p65 antibody (1: 2000, Abcam, ab16502); anti-β-actin antibody (1: 1000, Abcam, ab8226).
3
Quantification of MMP-9 Protein Levels
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The cytoplasmic proteins were prepared as we described before [30 (link)]. Briefly, hippocampal and cortical tissues were homogenized in RIPA buffer (Sigma-Aldrich, St. Louis, MO) containing protease inhibitor cocktail (10 mg/ml aproteinin, 5 mg/ml pepstatin, 5 mg/ml leupeptin, and 1 mM phenylmethanesulfonylfluoride) and placed on ice for 30 min. The homogenates were centrifuged at 13,000 rpm for 25 min at 4°C. The supernatant was collected for Western blotting. Protein concentration was determined by BCA assay.
The primary antibodies used were the rabbit polyclonal anti-MMP9 antibody (1:200 dilution, catalogue number: sc-10737; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit polyclonal anti-β-actin antibody (1:1000 dilution, catalogue number: 4967; Cell Signaling Technology Inc., Danvers, MA). The secondary antibody was goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (1:5000 dilution; Santa Cruz Biotechnology). The densities of MMP-9 protein bands were normalized to those of β-actin from the same sample.
The primary antibodies used were the rabbit polyclonal anti-MMP9 antibody (1:200 dilution, catalogue number: sc-10737; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit polyclonal anti-β-actin antibody (1:1000 dilution, catalogue number: 4967; Cell Signaling Technology Inc., Danvers, MA). The secondary antibody was goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (1:5000 dilution; Santa Cruz Biotechnology). The densities of MMP-9 protein bands were normalized to those of β-actin from the same sample.
4
Immunohistochemical Analysis of Tumor Markers
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For immunohistochemical analysis, isolated tumor tissues were fixed in 4% formamide, paraffin-embedded and sliced for H&E staining or immunohistochemical staining. For immunohistochemical staining, anti-Notch1 antibody (Enzo Life Sciences Inc, ADI-905-897, Farmingdale, NY, USA), anti-MMP-2 antibody (SC-10736; Santacruz Biotech. Inc.), anti-MMP-9 antibody (SC-21733; Santacruz Biotech. Inc.), and anti-proliferating cell nuclear antigen (PCNA) antibody (SC-25280; Santacruz Biotech. Inc.) were used at a dilution of 1:100 and the Envision kit (Life Technologies, Carlsbad, CA, USA) was used for the staining procedure.
5
Immunoblotting of Matrix Metalloproteinases
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The procedure was performed as previously used [19] (link). The following antibodies(Santa Cruz, USA) were used: anti-MMP-2 antibody (dilution 1∶150), anti-MMP-9 antibody (dilution 1∶200), anti-p50 antibody (dilution 1∶200), anti-p65 antibody (dilution 1∶200), anti-IκBα antibody(dilution 1∶300), anti-phospho-IκBα antibody(dilution 1∶200), anti-c-jun antibody (dilution 1∶200)), anti-jun-B antibody (dilution 1∶150), anti-jun-D antibody (dilution 1∶200), anti-c-Fos antibody (dilution 1∶200), anti-Fos-B antibody (dilution 1∶250).β-actin was used as internal control protein. Experiments were repeated at least three times.
6
Antibody Generation and Characterization
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A rabbit polyclonal anti-N-ChM-I antibody (N-ChM-I Ab) was raised against a synthetic oligopeptide (NH2-PSTTRRPHSEPRGNAGPGRLSNRTRP-CO2H) corresponding to the amino acids 8-33 of rat ChM-I [7] (link). A mouse monoclonal anti-ChM-I antibody (ChM-I MoAb) was raised in mice by immunizing recombinant human ChM-I expressed in 293-F cells [8] (link). The purified ChM-I MoAb (clone: hCHM-05) is commercially available from Cosmo Bio Co., Ltd. (Tokyo, Japan). Anti-CD31 antibody was obtained from BD PharMingen (San Diego, CA). Anti-MMP9 antibody was obtained from Santa Cruz Biotechnology (Dallas, TX). Anti-MMP13 antibody was from Merck Millipore (Billerica, MA). Anti-β-actin antibody (AC-15) was from Sigma.
7
Protein Expression Analysis in Kidney Tissue
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The kidney tissue was homogenized in protein lysate buffer (Leagene, PS0013, China). The supernatant was harvested after centrifugation at 4°C, and the protein was quantified using a bicinchoninic acid assay kit (Leagene, PT0006, China). The proteins were separated by gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Leagene, PE0017, China) and electrophoretically transferred to polyvinylidene difluoride blotting membranes (GE, 10600023, USA). The membranes were blocked with 5% non-fat dry milk for 2 h, and subsequently incubated with primary antibodies overnight at 4°C. The primary antibodies used were as follows: anti- MMP-9 antibody (1:500, Santa Cruz, sc393859), anti- PPAR-γ (1:500, Santa Cruz, sc7273), anti-NF-κB (1:500, SANTA, sc8008, USA) and anti-β-actin (1:5,000, Bioworld, BS6007M, USA). After being washed with TBST (Solarbio, T1081, China), the membranes were incubated with anti-mouse HRP labelled secondary antibodies (1:5,000, SeraCare, 5450-0011, USA). Signal quantification was performed using an Odyssey Infrared Imaging System (Li-COR, USA). The protein bands were normalized to the β-actin band in each sample.
8
Immunohistochemical Analysis of Renal MMP-9
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Paraffin-embedded kidney tissue slices (3 mm thickness) were deparaffinized. Then, the slices were treated with 3 % hydrogen peroxide in methanol for 10 min. Antigen retrieval was performed with citrate buffer for 5 min in a 100 °C water bath. The slices were then incubated with 10 % goat serum for 10 min at room temperature, followed by incubation with anti-MMP-9 antibody (1:500, Santa Cruz, CA, USA) at 4 °C overnight. Next, the slices were incubated with horseradish peroxidase for another 30 min. After incubation with secondary antibodies, the sites of peroxidase activity were carried out by 3, 3-diamino-benzidine tetrahydrochloride stain as a substrate, followed by hematoxylin staining. Every slide was examined MMP-9 stains in the normal and atrophic renal tubules.
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