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Tissue lysis extraction reagent

Manufactured by Merck Group
Sourced in United States, Switzerland

Tissue lysis/extraction reagent is a laboratory solution designed to facilitate the extraction and purification of biological molecules, such as DNA, RNA, and proteins, from tissue samples. The reagent functions by disrupting the cellular structure and releasing the intracellular contents, allowing for the subsequent isolation and analysis of the target biomolecules.

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13 protocols using tissue lysis extraction reagent

1

Protein Extraction from Prostatic Tissue

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Prostatic tissue was homogenized (1/10 w/v) in tissue lysis/extraction reagent (Sigma-Aldrich, St. Louis, MO, USA) containing protease inhibitor cocktail (Roche, Mannheim, Germany) using a homogenizer. Homogenates were centrifuged at 15,500× g for 20 min at 4 °C. Total protein concentrations in the supernatant fractions were measured using BCA protein reagent (Thermo Fisher Scientific, Waltham, MA, USA).
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2

Lung Tissue Protein Expression Analysis

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The lung tissue was homogenized (1/10 w/v) using a homogenizer in tissue lysis/extraction reagent (Sigma-Aldrich) supplemented with protease inhibitors (Sigma-Aldrich). Immunoblotting was performed as described previously [12 (link)]. The following primary antibodies were used: anti-PDE4B (1:1000 dilution; Abcam), anti-MMP-9 (1:1000 dilution; Abcam), and anti-β-actin (1:1000 dilution; Cell signaling, Denver, MA, USA). Expression of PDE4B, MMP-9, and β-actin were evaluated using ChemiDoc (Bio-Rad, Hercules, CA, USA).
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3

Lung Tissue Homogenization and Immunoblotting

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Homogenization of lung tissue was performed using a tissue lysis/extraction reagent (Sigma-Aldrich) with a protease inhibitor cocktail (Sigma-Aldrich). Immunoblotting was performed as previously described [14 (link)]. The primary antibodies were used as follows; anti-collagen (1:1,000 dilution; Santa Cruz), anti-TGF-β1 (1:1,000 dilution; Abcam), anti-β-actin (1:2,000 dilution; Cell Signaling, MA, USA) anti-pSmad 2/3 (1:1,000 dilution; Abcam) and anti-Smad 2/3 (1:1,000 dilution; Abcam). Protein expression value was determined by ChemiDoc (Bio Rad Lab.).
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4

Lung Protein Quantification and Analysis

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Lung tissues were homogenized with a tissue lysis/extraction reagent (Sigma-Aldrich, Carlsbad, CA, United States). The concentration of proteins in each sample was determined using Bradford reagent (Bio-Rad Laboratories, Hercules, CA, United States). Equal amounts of cellular proteins (30 μg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated in blocking solution (5% skim milk, Millipore Co., Bedford, MA, United States), followed by overnight incubation at 4°C with the appropriate primary antibodies, as follows: anti-p65 (Cell Signaling, Denver, MA, United States), anti-p-p65 (Cell Signaling) anti-MUC5AC (Abcam), anti-AP-1 (Cell Signaling) and anti-β-actin (Cell Signaling) antibodies. The blots were washed with Tris-buffered saline containing Tween 20 (TBST), and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA, United States) for 30 min at room temperature. The blots were washed again with TBST, and then, developed using an enhanced chemiluminescence kit (Thermo Scientific, San Diego, CA, United States). We evaluated the band expression values by ChemiDocTM (Bio-Rad, Hercules, CA, United States).
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5

Western Blot Analysis of Liver Proteins

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Liver tissue was homogenized (1:9, w/v) with a tissue lysis/extraction reagent (Sigma-Aldrich) containing a protease inhibitor cocktail (Sigma-Aldrich). Equal amounts of protein (40 µg/well) from each sample were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Whatman, Maidenstone, UK). The membrane was blocked with blocking buffer (5% skim milk) for 1 h at room temperature followed by an overnight incubation at 4℃ with appropriate primary antibodies. The following primary antibodies and dilutions were used: anti-Lamin B1 (1:1000 dilution; Santa Cruz Biotechnology, CA, USA), p38 MAPK, phospho-p38 MAPK (p-p38 MAPK), p44/42 MAPK (Erk1/2), p-Erk1/2, c-Jun amino-terminal kinase (JNK), p-JNK, iNOS, Cox-2, NF-κB p65, TNF-α, and β-actin (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA). The membrane was washed three times with Tris-buffered saline containing Tween 20 (TBST) and then incubated with horseradish peroxidaseconjugated secondary antibodies (1:2000 dilution; Sigma-Aldrich) for 1 h at room temperature. The blots were washed thrice with TBST and then developed using an enhanced chemiluminescence kit (Thermo Scientific, Waltham, MA, USA). To determine protein expression level, each band density was quantified using TINA 20 Image software (Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany).
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6

Lung Tissue Protein Analysis Protocol

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Protease and phosphatase inhibitors (Roche, Basel, Switzerland) were added to tissue lysis/extraction reagent (Sigma-Aldrich, St. Louis, MO, USA), and the lung tissue was homogenized with this solution (1/10 w/v). Protein concentrations of each sample were detected using BCA (Thermo Fisher Scientific, Waltham, MA, USA). Western blotting was performed as described previously [20 (link)], and the following antibodies were used: iNOS (1:000; Cell Signaling, Danvers, MA), COX-2 (1:000; Cell Signaling), NF-kB (1:000; 1:1000; ABCAm, Cambridge, UK), phosphor-NF-kB (1:1000; ABCAm, Cambridge, MA, USA) and MMP-9 (1:1000; ABCAm, Cambridge, MA, USA). Horse-radish peroxidase-conjugated secondary antibodies (1:10,000; Thermo Fisher Scientific, Waltham, MA, USA) were used and then detected with EZ-Western Lumi Pico (Dogenbio, Seoul, Republic of Korea). The densitometric values of each band were numerically expressed using Chemi-Doc (Bio Rad Laboratories, Hercules, CA, USA).
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7

Lung Tissue Protein and Signaling Analysis

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To identify the proteins and signaling pathways within the lung tissue, the right lung was homogenized with a tissue lysis/extraction reagent (Sigma-Aldrich). The total protein content of each sample was determined by using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). A total of 30 μg protein was electrophoresed by using 10% SDS-polyacrylamide gel and then transferred to a polyvinyl difluoride membrane. The membrane was blocked with 5% skim milk for 1 h and incubated overnight at 4 °C with the following primary antibodies: p-65 (diluted 1:1000, Abcam), phosphorylated-p65 (diluted 1:1000, Abcam), iNOS (diluted 1:1000, Abcam), HO-1 (diluted 1:1000, Cell Signaling, Beverly, MA, USA), Nrf2 (diluted 1:1000, Cell Signaling), β-actin (diluted 1:1000, Cell Signaling), lamine B1 (diluted 1:1000, Abcam). After washing three times with tris-buffered saline (TBS) that was supplemented with Tween 20, the horseradish peroxidase (HRP)-conjugated secondary antibody was diluted to 1:3000 and incubated for 1 h. Membranes were washed again, and binding was detected by using an enhanced chemiluminescence kit (Thermo-Scientific, Waltham, MA, USA). The density of each protein band was evaluated by using ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA).
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8

Western Blot Analysis of Lung Tissue

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The lung tissue was homogenized (1/10 w/v) using a homogenizer in a Tissue Lysis/Extraction reagent (Sigma-Aldrich, St, Louis, MO, USA) that contained a protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined using Bradford reagent (Bio-Rad). Equal amounts of the total protein (30 µg) were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were incubated with blocking solution (5% skim milk) followed by overnight incubation at 4℃ with the appropriate primary antibody. The following primary antibodies and dilutions were used: anti-β-actin (1:2000 dilution; Cell Signaling, Danvers, MA, USA), anti-pERK (1:1000 dilution; Cell Signaling), anti-ERK (1:1000 dilution; Cell Signaling) and anti-iNOS (1:1000 dilution; Santa Cruz Biotechnology, MA, USA). The blots were washed three times with Tris-buffered saline containing Tween 20 (TBST) and then incubated with a 1:10000 dilution of horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA, USA) for 30 min at room temperature. The blots were then washed three times with TBST and then developed using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Carlsbad, CA, USA).
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9

Western Blot Analysis of Prostate Tissue

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The prostate tissue was homogenized by using a homogenizer in a tissue lysis/extraction reagent (1/10 [w/v]; Sigma-Aldrich) containing a protease inhibitor (Roche, Basel, Switzerland). The protein concentration for each sample was determined by using the Bradford reagent (Bio-Rad Laboratories). Western blotting was performed as described previously [14 (link)] by using the following antibodies: anti-PCNA (1:1,000; Abcam, Cambridge, UK); anti-β-actin (Cell Signaling Technology, Beverly, MA, USA). To determine protein expression, the density of each band was quantified by using ChemiDoc (Bio-Rad Laboratories).
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10

Prostate Protein Expression Analysis

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Prostate tissue was homogenized with a tissue lysis/extraction reagent (Sigma‐Aldrich) and protein concentration was determined using Bradford reagent (Bio‐Rad Laboratories). Immunoblotting was performed according to methods described previously (Ko et al., 2018 (link)). Equal amounts of protein (30 μg) were heated at 100℃ for 5 min, loaded onto 10% SDS‐PAGE gels, and electrophoresed. The following primary antibodies and dilutions were used: anti‐PCNA (1:1,000 dilution, Abcam) and anti‐β‐actin (1:2000 dilution, Cell Signaling). To evaluate the proportion of protein expression, densitometric band values were determined using a Chemi‐Doc imaging system (Bio‐Rad Laboratories).
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