Urine samples were collected at the time of enrollment from all patients and immediately before hospital discharge from surviving patients who did not require permanent dialysis. Supernatants were frozen and stored at –70°C. TIMP-2, IGFBP7, and NGAL levels were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (TIMP-2, Abcam, Cambridge, UK; IGFBP7, Biorbyt, Cambridge, UK; NGAL, BioPorto, Gentofte, Denmark; and KIM-1, LSBio, Seattle, WA, USA). The amount of urine NGAL and KIM-1 excretion was expressed as ng/mL for comparison and normalized to the urine creatinine concentration (ng/mg creatinine).
Timp 2
Manufactured by Abcam
Sourced in United States, United Kingdom
TIMP-2 is a tissue inhibitor of metalloproteinases. It functions to inhibit the enzymatic activity of matrix metalloproteinases, which play a role in the breakdown of the extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Timp 1, by Abcam (16 mentions)
Mmp 9, by Abcam (6 mentions)
Pvdf membrane, by Merck Group (3 mentions)
β actin, by Abcam (3 mentions)
α sma, by Merck Group (3 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Collagen 1, by Abcam (2 mentions)
Collagen 3, by Abcam (2 mentions)
Criterion gel system, by Bio-Rad (2 mentions)
Caveolin 1, by Abcam (2 mentions)
Trans blot turbo system, by LI COR (2 mentions)
800cw or 680cw ir dye, by LI COR (2 mentions)
Odysseyxl system, by LI COR (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
P erk1 2, by Cell Signaling Technology (2 mentions)
Mmp 9, by Merck Group (2 mentions)
H3k27me3, by Cell Signaling Technology (2 mentions)
Tween 20, by Merck Group (2 mentions)
31 protocols using timp 2
1
Urine Biomarkers for Acute Kidney Injury
2
Western Blot Analysis of Cellular Proteins
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Cell extracts were prepared by lysis in 1× SDS loading buffer; then, the samples were boiled and subjected to Western blot analysis. Proteins (30 μg) were transferred from gels onto PVDF membranes (Millipore, Billerica, MA, USA) and blocked in 5% skimmed milk for 2 h. The following antibodies were used in the experiment to detect proteins: rabbit anti-SIRT1 (1:1000, Abcam, Cambridge, UK), EGR1 (1:1000, Abcam), USF2 (1:1000, Abcam), NF-κB p65 (acetyl K310) (1:1000, Abcam), TIMP-2 (1:1000, Abcam), MMP-2 (1:500, ProteinTech, Wuhan, China), MMP-9 (1:500, ProteinTech), mouse anti-β-actin (1:2000, Cell Signaling Technologies, Beverly, MA, USA), and NF-κB p65 (1:100, Santa Cruz, Dallas, Texas, USA). The antibodies were incubated with the membranes at room temperature for 1 h and then at 4 °C overnight. HRP-linked anti-rabbit and anti-mouse secondary antibodies were incubated with the membranes at 4 °C for 1 h. All blotting was detected by the enhanced chemiluminescence (ECL) detection method.
3
Protein Expression Analysis Workflow
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CM were mixed with 4× Laemmli sample buffer (containing 2-mercaptoethanol) and boiled for 10 min at 95°C. 15-μl protein volumes were separated on 4–15% Mini-PROTEAN TGX stain-free protein gels (4568086; Bio-Rad), and whole proteins were detected with the Criterion Stain-free imaging system (Bio-Rad). Next, the proteins were transferred on polyvinylidene difluoride membranes using the Trans-Blot-Turbo system (Bio-Rad). The membranes were blocked in 5% nonfat dry milk/TBS-T for 1 h at RT. The following primary antibodies were diluted 1:1,000 in 5% BSA/TBS-T and incubated overnight at 4°C: MMP1 (sc-30069; Santa Cruz), SERPINE1 (sc-5297; Santa Cruz), TIMP2 (ab53730; Abcam), COL6A1 (sc-377143; Santa Cruz), FN1 (F3648; Sigma-Aldrich), and THBS1 (sc-59887; Santa Cruz). Membranes were 3× washed in TBS-T and the following secondary antibodies were diluted 1:20,000 in 2.5% nonfat dry milk/TBS-T and incubated for 1 h at RT: goat-anti-mouse-HRP (SAB3701073-2; Sigma-Aldrich) or goat-anti-rabbit-HRP (SAB3700878-1; Sigma-Aldrich). HRP was visualized by the UltraScence Pico Ultra Western Substrate (CCH345-B; GeneDireX) and the ChemiDoc MP imaging system (Bio-Rad).
4
Antibody Sourcing for Western Blot
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Antibodies specific to cleaved caspase-3, COX-2, p-IKKβ, IKKβ, p-IκBα, IκBα, p65, β-actin and all the secondary antibodies were purchased from Cell Signaling Technology (Cell Signaling Technology, Inc., USA). Antibodies specific to cytochrome c, and p50 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies specific to Bcl-2, BAX, and Lamin B1 were purchased from Proteintech Group (Proteintech, Inc., USA). Antibodies specific to MMP-2/9 and TIMP-2 were purchased from Abcam. RPMI-1640 media, fetal bovine serum (FBS), and trypsin were purchased from Gibco. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise specified.
5
Rabbit Aorta and VSMC Protein Analysis
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Total protein was extracted from the aorta of rabbits and VSMCs as previously described [22 (link)]. The primary antibodies were against rabbit's DDR2 (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA), rat's DDR2 (1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA; CST, Beverly, MA, USA), MMP-2 (1 : 500; Abcam, Cambridge, MA), TIMP-1 (1 : 500; Abcam, Cambridge, MA), TIMP-2 (1 : 500; Abcam, Cambridge, MA), p-ERK1/2 (1 : 1000; CST, Beverly, MA, USA), and GAPDH (1 : 1000; Santa Cruz Biotechnology, Santa Cruz, CA). Western blotting analysis was applied as previously described, and relative protein expression was measured by ImageJ with gel analysis [22 (link)].
6
Quantitative Western Blot Analysis
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A 50-microgram protein was loaded on SDS–PAGE, and then transferred to nitrocellulose sheets (NEN Life Science Products, Inc., Boston, Massachusetts). After blocking, the blots were incubated with MMP-2, MMP-9, TIMP-2, or β-actin antibody (Abcam, Cambridge, United Kingdom) (dilution 1:1000) in 5% non-fat skimmed milk (using β-actin as a loading control). After the blots were washed, they were incubated with secondary antibodies conjugated with alkaline phosphatase (dilution 1:3000) (Jackson ImmunoResearch Laboratories Inc., Philadelphia, Pennsylvania). Immunoblots were developed using bromochloroindolyl phosphate/nitroblue tetrazolium solution (Kirkegaard and Perry Laboratories Inc., Baltimore, Maryland).
7
Quantitative Analysis of Cell Signaling
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Whole cell lysates or concentrated conditioned media were separated on SDS-PAGE gels (7%, 10%, or 4-15% gradient gels, Criterion Gel System; Bio-Rad, Hercules, CA) via standard techniques. Proteins were transferred to nitrocellulose membranes using a Bio-Rad Trans-Blot Turbo system and blocked in Odyssey Blocking buffer (Li-Cor Biosciences, Lincoln, NE) prior to overnight incubation in a 1μg/mL solution of primary antibody of interest. Primary antibodies included: caveolin-1, collagen I, collagen III (Abcam, Cambridge, MA), Cavin-1, Cavin-3, fibronectin, matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9), tissue inhibitor of metalloproteinase 1 (TIMP1), and tissue inhibitor of metalloproteinase 2 (TIMP2) (Santa Cruz Biotechnology, Santa Cruz, CA). Secondary antibodies were conjugated to 800CW or 680CW IR dye (Li-Cor Biosciences) and imaging was performed using a Li-Cor OdysseyXL system prior to quantification via densitometry. GAPDH expression was used to normalize cell lysate protein concentrations. Concentrated conditioned media was normalized to cell lysate protein concentrations as determined by Lowry protein assays.
8
Histological Assessment of Kidney Injury
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Tubular injury was assessed using periodic acid-Schiff (PAS)-stained kidney sections. For immunohistochemical staining, we used rat anti-mouse F4/80 (Serotec, Kidlington, UK), Gr-1 (eBioscience, San Diego, CA, USA), CSF-1 (Abcam, Cambridge, UK), TIMP-2 (Abcam), pH3 (Abcam) antibodies. A total of 8–10 high power fields (HPFs) were captured, and the mean number of positive cells was compared between groups.
9
Comprehensive Antibody Panel for Cellular Signaling Investigations
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Antibodies to EZH2, H3K27me3, RKIP, cleaved Caspase-3, and P-ERK1/2 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to E-cadherin, MMP-2, P-Raf-1, and GAPDH were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cleaved PARP (24 kDA) and TIMP-2 antibodies were purchased from Abcam, Inc. (Cambridge, MA). Neutrophil gelatinase-associated lipocalin NGAL antibody was obtained from R&D Systems (Minneapolis, MN). ZO-1, MMP-9, TIMP-3 antibodies were purchased from EMD Millipore (Billerica, MA). 3-DZNeP was purchased from Selleck Chemicals (Houston, TX). ɑ-tubulin and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). SiRNA specific for EZH2 was obtained from Invitrogen (Carlsbad, CA). All other antibodies used in this study were purchased from Cell Signaling Technology (Danvers, MA).
10
Western Blot Analysis of TIMP and MMP
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For the detection of TIMP and MMP protein expression 15 μg of total protein was separated on a 10 % polyacrylamide gel and transferred to Immobilon-P membranes (Millipore, Schwalbach, Germany). After blocking in PBS supplemented with 5 % skim milk (Sigma-Aldrich) and 0.05 % Tween 20 (Sigma-Aldrich) membranes were incubated overnight at 4 °C with one of the following primary antibodies at the indicated dilutions: MMP2 (1:500) (Abcam, Cambridge, United Kingdom), MMP9 (1:500) (Abcam) TIMP1 (1:250) (Abcam), TIMP2 (1:250) (Abcam), TIMP3 (1:250) (Abcam). After washing three times in PBS containing 0.1 % Tween 20 membranes were incubated for 1 h at room temperature with 5000-fold diluted peroxidase conjugated goat anti-mouse IgG (Santa Cruz). Proteins recognized by the antibody were visualized with luminata forte western blotting substrate (Millipore) according to the manufacturer’s instructions. Signal intensities were quantified by densitometry using Bio-1D software version 15.01 (Vilber Lourmat, Eberhardzell, Germany).
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