Total protein lysates were prepared from cultured cells or tissue samples with modified Tissue Protein Extraction Reagent (TPER) (Life Technologies Corporation, Carlsbad, CA, USA) supplemented with 300 mM NaCl and a cocktail of protease and phosphatase inhibitors as previously described (Roche, Pleasanton, CA, USA) [24 , 25 (link), 28 (link)].
Tissue protein extraction reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, India, United Kingdom
Tissue Protein Extraction Reagent is a solution designed for the isolation and purification of proteins from tissue samples. It facilitates the efficient extraction of a wide range of proteins, including both soluble and insoluble proteins, from various types of tissue.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Protease inhibitor cocktail, by Merck Group (8 mentions)
Las 4000, by Cytiva (7 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (4 mentions)
Sirt5, by LifeSpan BioSciences (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor, by Roche (4 mentions)
Protease inhibitor, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Complete mini, by Roche (3 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (3 mentions)
Bip grp78, by Abcam (3 mentions)
Las4000 imager, by GE Healthcare (3 mentions)
Sirt7 and foxo1 acetyl lys294, by LifeSpan BioSciences (3 mentions)
151 protocols using tissue protein extraction reagent
1
Extracting Tissue Protein Lysates
2
Aβ and NfL Quantification in Brain Tissue
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Sample preparation and quantification of Aβ was performed as described.
33 (link),
35 (link) Brain hemispheres were microdissected and separated into cortical and hippocampal tissues, then flash‐frozen for biochemical analysis. The samples were pulverized using a Bessman Tissue Pulverizer kit, and half of pulverized tissue was homogenized in 1000 μL/150 mg (cortex) or 150 μL (hippocampus) Tissue Protein Extraction Reagent (TPER; Life Technologies, Grand Island, NY, USA) containing protease (Roche, Indianapolis, IN, USA) and phosphatase inhibitors (Sigma‐Aldrich) before centrifugation at 100,000 × g for 1 h at 4°C to generate TPER‐soluble fractions. For formic‐acid fractions, pellets from the TPER‐soluble fraction were homogenized in 500 μL (cortex) or 75 μL (hippocampus) 70% formic acid followed by centrifugation at 100,000 × g for 1 h at 4°C. Protein concentration was calculated via Bradford Protein Assay. Human Aβ in soluble and insoluble fractions was quantified using the V‐PLEX Aβ Peptide Panel 1 (6E10) (K15200G‐1; Meso Scale Discovery, Rockville, MD, USA) and the neurofilament light chain (NfL) in plasma and cortex was quantified using the R‐Plex Human Neurofilament L Assay (K1517XR‐2; Meso Scale Discovery), all according to the manufacturer's instructions.
33 (link),
35 (link) Brain hemispheres were microdissected and separated into cortical and hippocampal tissues, then flash‐frozen for biochemical analysis. The samples were pulverized using a Bessman Tissue Pulverizer kit, and half of pulverized tissue was homogenized in 1000 μL/150 mg (cortex) or 150 μL (hippocampus) Tissue Protein Extraction Reagent (TPER; Life Technologies, Grand Island, NY, USA) containing protease (Roche, Indianapolis, IN, USA) and phosphatase inhibitors (Sigma‐Aldrich) before centrifugation at 100,000 × g for 1 h at 4°C to generate TPER‐soluble fractions. For formic‐acid fractions, pellets from the TPER‐soluble fraction were homogenized in 500 μL (cortex) or 75 μL (hippocampus) 70% formic acid followed by centrifugation at 100,000 × g for 1 h at 4°C. Protein concentration was calculated via Bradford Protein Assay. Human Aβ in soluble and insoluble fractions was quantified using the V‐PLEX Aβ Peptide Panel 1 (6E10) (K15200G‐1; Meso Scale Discovery, Rockville, MD, USA) and the neurofilament light chain (NfL) in plasma and cortex was quantified using the R‐Plex Human Neurofilament L Assay (K1517XR‐2; Meso Scale Discovery), all according to the manufacturer's instructions.
3
Isolation of Soluble and Insoluble Protein Fractions
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To isolate protein for the ELISA, flash-frozen brain hemispheres were microdissected into cortical, hippocampal, and thalamic regions and grounded to a powder. Hippocampal tissue was then homogenized in Tissue Protein Extraction Reagent (TPER (Life Technologies, Grand Island, NY)) with protease and phosphatase inhibitors present. Samples were centrifuged at 100,000 g for 1 h at 4 °C to generate TPER-soluble fractions. To generate formic acid fractions, protein pellets from the TPER-soluble fraction were then homogenized in 70% formic acid and centrifuged at 100,000 g for 1 h at 4 °C, the formic acid fraction is then neutralized. Quantification of soluble and insoluble fractions of both Aβ and NfL was performed as previously described [67 (link)].
4
Protein Isolation and Quantification from Brain Tissues
5
Lung Cytokine Quantification Protocol
6
Cytokine Profile Analysis of Skin Samples
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Tissue Protein Extraction Reagent (Thermo Fisher Scientific) was used to extract protein from crushed skin samples. For 20 min, the proteins were centrifuged at 12,000× g. The supernatant was then collected, and the protein contents were determined using the Erba total protein assay kit. Commercially available Invitrogen/Thermo Fisher Scientific, enzyme-linked immunosorbent assay (sandwich ELISA) kits (Thermo Fisher Scientific India Pvt. Ltd., Powai, India) were used for IL-22 (IL-22 Mouse ABTS ELISA Development Kit), IL-6 (IL-6 Mouse ELISA Kit), IL-17 (IL-17A Mouse ELISA Development Kit ABTS), TNF-α (TNF alpha Mouse ELISA Kit, Elabscience) IL-1β (IL-1β Mouse ELISA Development Kit ABTS) and MCP-1/CCL2 (mouse MCP-1 ELISA kit, FineTest). All tests were performed as per the manufacturer’s instructions manual [15 (link)].
7
Western Blot Analysis of Esophageal Tissue
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Frozen rat esophagus tissue was homogenized in Tissue Protein Extraction Reagent (ThermoFisher, Waltham, MA, USA) using TissueLyser II (Qiagen) at 30 Hz for 5 min. Extracted protein was then quantified using the DC Protein Assay (Bio-Rad). Western blot analysis was performed as previously described [123 (link)]. Images were captured using the Bio-Rad ChemiDoc Imaging System and quantified by means of chemiluminescent immunodetection using Bio-Rad Image Lab Software version 6.1.0 with expression levels normalized to the loading control GAPDH. Immunoblotting was performed using commercially available antibodies from Abcam (Cambridge, MA, USA) and Cell Signaling Technology (Danvers, MA, USA): ABST (ab203205, 1:500), ATF-4 (CST #11815, 1:1000), CD44 (ab189524, 1:500), GAPDH (CST #2118, 1:25,000), HSP60 (CST #12165, 1:5000), IRE1α (CST #3294, 1:1000), and phospho-AMPK (CST #2535, 1:1000). Patient EAC samples with matched normal and BE tissues were collected at the University Hospital at the University of Michigan. Informed consent was obtained from patients prior to sample collection. Protein extraction and quantification were similarly performed as described above.
8
Quantification of Sp1, Sp3, and SOD2 in HIV-Infected Mouse Lungs
9
Protein Extraction from Rat Organs
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A section of the left lobe of the liver (approximately 1.1 g), half of the spleen, one kidney, and one lung were collected and placed in 600 μL of the tissue-protein extraction reagent (Thermo Fisher) immediately after removal from the rat and kept on ice. Organs were homogenized using ceramic beads (Mobio) in a TissueLyser (QIAGEN). Tissue homogenates were incubated on ice for 30 min and then centrifuged to remove tissue debris. Total protein in the lysate was determined by the bicinchoninic acid assay kit (Thermo Fisher) according to the manufacturer's directions using bovine serum albumin as the standard. HBP concentrations in rat organ lysates were normalized to total protein levels.
10
Immunoblotting Analysis of Cell Lysates
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Cell lysates were prepared using a tissue protein extraction reagent (Thermo Fisher Scientific). Immunoblotting analysis was carried out on the lysates, which were separated by SDS‐PAGE and transferred to Immobilon‐P membranes (Merck Millipore). The membranes were incubated in PVDF blocking reagent for Can Get Signal immunostain (Toyobo, Osaka, Japan) at room temperature for 1 h to block non‐specific reactions, overnight at 4°C with primary antibodies at the appropriate concentrations (see Reagents ). Then, HRP‐conjugated secondary antibodies were applied to the membrane for 1 h and photographs of the image were taken on LAS‐3000 (Fujifilm, Tokyo, Japan) using ECL prime Western blotting detection reagent (GE Healthcare, Chicago, IL, USA).
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