Pulverized 3D-LTCs were lysed in T-PER lysis buffer (Thermo Fisher Scientific, Germany) containing proteinase and phosphatase inhibitors (Roche, Switzerland). Protein concentration was assessed using the BCA assay (Thermo Fisher Scientific, Germany) according to the manufacturer’s instructions. 15 μg of total protein was separated on SDS-polyacrylamide gels and transferred to PVDF membranes (Biorad, USA). The membranes were blocked in 5% nonfat dry milk (Applichem, Germany) and incubated with the primary antibody (at 4 °C overnight followed by 1 h at RT). Subsequently the blots were incubated with respective secondary, HRP-conjugated, antibody (GE-Healthcare) for 1 h, washed and visualized using chemiluminescence reagents (Pierce ECL, Thermo Fisher Scientific, Germany) with the ChemiDocTMXRS+ system. Analysis of secreted collagen was performed by concentrating 200 μl of supernatant from the same number of 4-mm punches generated from 3D-LTCs in each group using Nanosep 10 K OMEGA columns (Pall Corporation, MI, USA) followed by dilution in 60 μl lysis buffer and as previously described [25 (link)].
T per lysis buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, France, Sweden
T-PER lysis buffer is a protein extraction reagent designed for the efficient extraction of proteins from tissue samples. It is a detergent-based buffer that aids in the solubilization and recovery of proteins from various tissues. The core function of T-PER lysis buffer is to facilitate the extraction of proteins from tissue samples for further analysis and characterization.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Pierce ecl, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Odyssey scanner, by LI COR (3 mentions)
Bradford protein assay, by Bio-Rad (3 mentions)
M per lysis buffer, by Thermo Fisher Scientific (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Chemidoc imaging system, by Bio-Rad (3 mentions)
Image lab software, by Bio-Rad (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Ipvh00010, by Merck Group (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Bradford assay kit, by Thermo Fisher Scientific (2 mentions)
Polytron tp 20 homogenizer 8, by Kinematica (2 mentions)
C digit blot scanner, by LI COR (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Phosphostop, by Roche (2 mentions)
Goat anti β actin, by Abcam (2 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
54 protocols using t per lysis buffer
1
Western Blot Analysis of 3D-LTC Proteins
2
Immunoblotting Analysis of Cardiac Proteins
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Protein extracts were prepared from cells and human left ventricular tissue using M-PER and T-PER lysis buffer (Thermo Scientific, NY) respectively [24 (link)]. Immunoblotting was performed using primary antibody specific for CTGF (Santa Cruz, CA), phospho-ERK1/2, Total-ERK1/2 (Cell Signaling, MA) and GAPDH (Ambion, IL) followed by incubation with infrared dye (IRDye)-conjugated secondary antibodies (LI-COR, NE) and immunoreactive bands were visualized under Odyssey scanner (LI-COR, NE). The bands were quantitated using Image Studio Ver3.1 (LI-COR, NE) and normalized by GAPDH.
3
Western Blot Quantification of Glutamate Transporters
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Finally, we measured the protein levels of glutamate transporters GLT‐1 and xCT in the NAc using Western blot. For this, proteins from NAc were extracted using T‐PER lysis buffer (Thermo‐Fisher) supplemented with a protease inhibitor. 25 μg of protein were loaded into each lane. The GLT‐1 protein was identified using a guinea pig anti‐GLT‐1 primary antibody (Cat AB1783, Millipore, 1:500 dilution) and an IRDye 800CW donkey anti‐guinea pig secondary antibody (Cat 925‐32411, LI‐COR, 1:10,000 dilution). For xCT detection, a rabbit anti‐xCT primary antibody (Cat AB175186, Abcam, 1:500 dilution) was used in conjunction with an IRDye 800CW donkey anti‐rabbit secondary antibody (Cat 926‐32213, LI‐COR, 1:10,000 dilution). β‐actin, detected using a mouse anti‐β‐actin primary antibody (Cat sc‐47778 Santa Cruz Biotechnology, 1:200 dilution) and an IRDye 800CW goat anti‐mouse secondary antibody (Cat 926‐32210, LI‐COR), served as the loading control. The reactive bands were captured with the Odyssey Imaging System (LI‐COR), and the intensities were quantified using Image Studio Lite 5.2 software.
4
Analyzing SOD1 Protein Turnover
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NSC34 cells (1.5×105 cells/well) were seeded into 12-well plates and transfected with one of the plasmids expressing FLAG-tagged A4T-SOD1, HA-tagged SUMO 1/3, or the empty vector and myc-tagged Ubc9 using lipofection. After 16 h of transfection, the cells were treated with 50 µg/ml cycloheximide (CHX) for 3 or 6 h and lysed in T-PER lysis buffer (Thermo Scientific) with phosphatase and protease inhibitors (Thermo Scientific). The cell debris was removed by centrifugation, and the clarified cell lysates (20 µg of protein) were subjected to an immunoblot analysis as described above. The band intensity was quantified using ImageJ (NIH), and the arbitrary units of SOD1 were normalized to those of β-actin.
5
Western Blot Analysis of Liver Proteins
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The proteins were extracted from the mouse liver tissues using T-PER lysis buffer (78510, Thermo Scientific, New York, NY, USA) with a protease inhibitor cocktail. Proteins were extracted from the cells using M-PER lysis buffer with a protease inhibitor cocktail. All of the proteins were quantified using a BCA protein assay kit. Protein samples were separated on suitable concentration SDS-PAGE gels and transferred to PVDF membranes (IPVH00010; Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in TBST, and then incubated with the indicated primary antibodies overnight at 4 °C. After being washed with TBST, the membranes were incubated with secondary horseradish peroxidase (HRP)-conjugated antibodies for 1 h at room temperature. Protein expression signals were detected using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). Beta-actin (ACTB) was used as the equal loading control. All of the antibodies used in this study are listed in Supplementary Table S2 .
6
Western Blotting Protein Detection Protocol
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Cells were lysed with T-PER lysis buffer supplemented with Halt protease and phosphatase inhibitor cocktails (all from Thermo Fisher Scientific). Lysed cells were sonicated in just 1 cycle of 15 s duration and at a power of 40–50%. Whole cell lysates were quantified with the Bradford Protein Assay (Bio-Rad). 40 µg of total proteins were separated on bolt 4–12% polyacrylamide-0.1% commercial SDS gels (Thermo Fisher Scientific) and transferred onto nitrocellulose membrane. Membranes were washed with PBS-0.1% Tween-20 (PBST) and non-specific binding sites were blocked with blocking buffer (5% w/v non-fat dry milk in PBST) for 1 h at room temperature. Lastly, membranes were incubated with primary antibodies overnight at 4 °C. The antibodies used were diluted 1:1000 in either bovine serum albumin (BSA) (Sigma-Aldrich) or milk following manufacturer’s protocols. Membranes were washed again with PBST, then incubated with peroxidase conjugated secondary antibodies (Thermo Fisher Scientific) diluted in PBST for 1 h at room temperature. ECL enhanced chemiluminescence reagents (Thermo Fisher Scientific) were used to detect immunoreactive bands and images captured with the ChemiDoc-It® 2Imager digital system (UVP, Upland, CA US).
7
Quantifying Neuroinflammatory Markers in Co-Cultures
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Conditioned media from the cell samples were collected immediately before protein extraction and centrifuged at 10000×g for 10 min. Aβ40 levels in the conditioned media were determined with monoclonal and HRP-conjugated antibody-based Human/Rat β amyloid 40 ELISA kit (Wako, Osaka, Japan). Aβ40 concentrations were normalized to neuronal viability. Mouse TNF alpha ELISA Ready-SET-Go! kit (Affymetrix, San Diego, CA, USA) was used for the detection of tumor necrosis factor α (TNFα) in the conditioned media. Nitric oxide (NO) levels were determined using Griess Reagent Kit for Nitrite Determination (G-7921, Life Technologies) and normalized to neuronal viability determined by the MAP2-ABTS assay described above. All kits were used as instructed by the manufacturers. Reactive oxygen species (ROS) levels in the co-cultures were measured using fluorogenic probe 2′, 7′-Dichlorodihydrofluorescin diacetate (DCFH-DA, Sigma D6883). One hour after adding BV2 cells, samples were labeled with 120 μM DCFH-DA for 30 min. Two hours after adding the BV2 cells, 5 h-neuroinflammation treatment was started. Subsequently, cells were lysed using T-PER lysis buffer (Thermo Scientific), and fluorescence was measured using plate reader at 480 nm/530 nm.
8
Protein Isolation from Porcine AV Leaflets and Human VICs
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After 1 week in culture, porcine AV leaflets were harvested for protein isolation as described previously (17 (link), 18 (link)). Briefly, the leaflets were flash frozen with liquid nitrogen and stored at −80°C overnight before being lyophilized for 24 h. The lyophilized tissues were harvested in T-PER lysis buffer (Thermo Fisher Scientific) containing 1% Halt™ protease inhibitor cocktail (Thermo Fisher Scientific) and homogenized using a Tissue Lyser II (Qiagen, Germantown, MD). The homogenized tissue lysates were incubated at 4°C for 1 h, then centrifuged at 10,000 × g for 10 min, and the supernatant was collected and stored at −80°C. Likewise, after 1 week in culture, cell lysates from human VICs were collected as described previously (19 (link), 20 (link)). The protein content was determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific) (20 (link)).
9
Quantifying SCF Levels in Murine Tissues
10
Protein Extraction from BIAHR and H
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The protein extraction was performed as previously described7 (link). Briefly, BIAHR and H proteins were solubilized with a commercially available T-PER lysis buffer (Thermo Fisher Scientific, France) complemented with a protease inhibitors cocktail (Thermo Fischer scientific, France). Solubilization was performed using a Polytron TP-20 Homogenizer 8 (Kinematica, Lucerne, Switzerland). After two centrifugations, at 13,000 g for 45 min at 4 °C to discard insoluble material from the supernatant, the protein concentration of every soluble extract was determined using a Bradford assay kit according to the manufacturer’s instructions (Thermo Fisher Scientific, France).
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