APEX proximity labeling was performed as previously described61 (link),62 (link). In brief, 80–90% confluent cells were pretreated with 500 μM biotin-tyramide for 30 min, followed by 1 mM H2O2 for 1min. After quenching and washing with PBS containing 5 mM Trolox and 10mM sodium ascorbate, cells were harvested into PBS. The cells were centrifuged, and cell pellets were lysed in RIPA buffer with 1 mM PMSF, 5 mM Trolox, 10 mM sodium ascorbate, and 10 mM sodium azide. Cell lysates were incubated with prewashed Pierce streptavidin magnetic beads (Thermo Fisher) overnight in a cold room. The beads were washed with RIPA buffer (Teknova) twice, with 1 M KCl, 0.1 M Na2CO3, and 2 M urea in 10 mM Tris-HCl (pH 8.0) once, and with RIPA buffer twice. Bound biotinylated proteins were eluted in 1 x SDS sample buffer and separated on 4–20% PAGE, followed by immunoblotting with NDUFS4 antibody. The immuno-precipitated samples were resolved on NuPAGE 10% Bis-Tris Gel. Each lane was excised into 4 equal pieces and combined into two tubes after in-gel digestion using LysC and trypsin enzymes. Digested peptide was processed and used for the LC-MS/MS analysis in the same way as complexsome profiling.
Ripa buffer
Manufactured by Teknova
Sourced in United States
RIPA buffer is a widely used cell lysis and protein extraction reagent. It is a solution containing detergents, salts, and buffers that helps to solubilize and extract proteins from cells and tissues. The buffer is designed to preserve the native conformation and activities of the extracted proteins.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Gradient sds page, by Bio-Rad (4 mentions)
Dc protein assay, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Trizol solution, by Thermo Fisher Scientific (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Anti e cadherin, by BD (2 mentions)
Anti vimentin, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Novus Biologicals (2 mentions)
Anti ck2α α, by Santa Cruz Biotechnology (2 mentions)
Ne per kit, by Thermo Fisher Scientific (2 mentions)
Ecl system, by Cytiva (2 mentions)
Anti gfp, by Santa Cruz Biotechnology (2 mentions)
Anti ck2β, by Thermo Fisher Scientific (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Anti flag m2 magnetic bead, by Merck Group (2 mentions)
45 protocols using ripa buffer
1
APEX Proximity Labeling for Proteomic Analysis
2
TFEB Transcriptional Activity Assay
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TFEB transcriptional activity was measured using Human TFEB Activity Assay Kit (RayBiotech) according to the manufacturer’s instruction. Cell lysates were prepared in RIPA buffer (TEKnova) supplemented with Protease /Phosphotase Inhibitor (Cell Signaling), followed by sonication. Protein concentration was measured using MicroBCA Protein Assay kit (Thermo Scientific). TFEB assay was performed in duplicate wells using 30–60 μg total protein per well and measured using a plate reader.
3
Conjunctival Impression Cytology Protocol
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Conjunctival impression cytology (CIC) was performed as per the protocol published [8 (link)]. The filter paper discs were peeled off and immediately placed in either 500 μL Trizol solution (Invitrogen, CA, USA) for RNA isolation or 100 μL of radio immunoprecipitation assay (RIPA) buffer (Teknova, CA, USA) for protein isolation.
4
Quantifying Phosphorylated HSL Levels
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Protein was extracted in RIPA buffer (Teknova, Hollister, CA) containing protease (Roche, San Francisco, CA) and phosphatase (Thermo Scientific, Waltham, MA) inhibitors and quantified using BCA reagent as described in [21 (link)]. Western blots were performed using the size-based Simple Western system (ProteinSimple, Santa Clara, CA) that automatically processes protein samples by capillary technology. Antibodies were from Cell Signaling Technology (Danvers, MA) and included rabbit anti-mouse hormone sensitive lipase (HSL, 1:100, #4107), rabbit anti-mouse phosphorylated HSL serine 660 (pHSL; 1:50, #4126), and rabbit anti-human vinculin as loading control (1:1,000, #4650). Digital images of the Western blots were analyzed with Compass software (ProteinSimple, Santa Clara, CA), and the quantified data for HSL and pHSL was normalized to vinculin. Data are reported as the ratio of pHSL:HSL as previously reported by [21 (link)].
5
Chloroquine Treatment and Protein Extraction
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Where indicated, cells were treated with 100 μM chloroquine overnight (Invitrogen, L10382- component B) before lysis. Samples were lysed in RIPA buffer (TEKnova, R3792) that had 1x mini EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, 11836170001) and Phosphatase Inhibitor Cocktail 2 and 3 (Sigma-Aldrich, P5726-5ML; P0044-5ML). Cells were sonicated on ice and centrifuged at 4°C at 20,000 g for 20 min, followed by a BCA assay (Thermo Fisher Scientific, 23225) for protein concentrations.
6
Immunoblotting Analysis of CSFV Infection
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For immunoblotting analysis infected cell monolayers or media only control (mock infected) were washed in ice-cold PBS and lysed in RIPA Buffer (Teknova, Hollister, CA, USA) in the presence of a protease inhibitor cocktail (Roche, Basel, Switzerland). Proteins were resolved on NuPAGE 4–12% w/v Bis-Tris gels (Invitrogen, Carlsbad, CA, USA) and transferred to polyvinylidene difluoride (PVDF) membranes following manufacturer instructions. Immunodetection was performed using the following antibodies: Rabbit polyclonal Anti-PPP1CB using a dilution of 1:1000 (Cat# ab53315, Abcam, Cambridge, UK), mouse monoclonal antibody anti-α-Tubulin using a dilution of 1:3000 DM1A (Thermo Fisher Scientific, Waltham, MA, USA), and anti-CSFV E2 protein monoclonal antibody WH303 using a dilution of 1:1000 [32 (link)] and Pierce Goat Anti-Rabbit IgG peroxidase conjugated or Pierce Goat Anti-Mouse IgG peroxidase conjugated secondary antibody reagent (Thermo Fisher Scientific, Waltham, MA, USA). Western blots were imaged using an Azure C300 and analyzed with cSeries capture software (Azure Biosystems, Dublin, CA, USA).
7
Protein Extraction and Western Blotting
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Proteins from tissues and cell lines were obtained as formerly described [30 (link)]. Protein extracts from human islets were freshly obtained either from Dr. Patrick MacDonald or after sonication of freshly isolated primary human (Prodo Labs, Table 2 )/mouse islets on ice by using RIPA buffer (Teknova, Hollister, CA) supplemented with protease/phosphatase inhibitor cocktails (ThermoFisher, Sci.). Up to 75μg of total proteins were loaded onto pre-casted 4–20% Tris-HEPES protein gels (Thermo Scientific-Pierce) or Bolt 4–12% Bis-Tris Plus gels (Invitrogen/ThermoFisher Sci.), run under denaturing conditions and electro-transferred onto PDVF membranes at 4°C by using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) or the iBlot-2 Gel Transfer Device (Invitrogen). After blocking overnight with 4% BSA, membranes were incubated with suitable primary antibodies (overnight at 4°C) and developed with HRP-conjugated secondary ones (2h at room temperature). We used ChemiDoc™ MP Imaging System with Image Lab™ Software (Bio-Rad, Berkeley, CA) to detect antigen-antibody reactions. High-resolution gray-scale digital images taken by using this imaging system were cropped to exclude irrelevant lanes and used without further modification. Original blots used to construct Figures are shown in S4 Fig .
8
Protein Expression Analysis in Kidney Cells
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Kidney tissues were homogenized and HK-2 cells were sonicated in RIPA buffer (TekNova, CA, U.S.A.). After centrifugation, supernatants were dissolved in Laemmli Buffer. The dissolved proteins were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, MA, U.S.A.). The membranes were blocked with 5% nonfat milk for 1 h and then were incubated overnight at 4°C with the following primary antibodies: Bcl-2, Bax (Cell Signaling, Danvers, MA, U.S.A.), ELA (AbboMax, San Jose, CA), or GAPDH (Proteintech, Chicago, IL, U.S.A.). After washing, membranes were incubated with the appropriate secondary antibodies and developed by enhanced chemiluminescence (Luminata, Millipore, Billerica, MA, U.S.A.). Protein bands were imaged and quantified by using Alpha View-Fluor ChemQ software (Alpha Innotech Gel Imaging System, San Jose, CA).
9
Protein Expression Analysis in Immortalized Cells
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