For protein extraction cells were lysed and sonicated or tissues were homogenized in ice-cold 1X RIPA buffer (Santa Cruz Biotechnology) containing protease and phosphatase inhibitor (Roche Applied science). Lysates were incubated on ice for 30 min and centrifuged at 10,000 × g for 10 min at 4°C. Supernatant fluid was collected and used as a total cell lysates for protein assays. Samples were stored at −80°C until further use. Protein concentration was measured by BCA protein assay and spectrophotometry at A280 (Nanodrop 2000, Thermo Scientific). Equal amounts of protein (5–20 μg) from total cell lysates were separated by 4–12% SDS-PAGE and blotted onto PVDF transfer membranes. The membranes were blocked at room temperature for 1 h in 5% nonfat dry milk in TBS-T. Blots were incubated with primary antibody to ATG3, p62, LC3B, LAMP-1, LAMP-2 and/or GAPDH overnight at 4°C and with appropriate secondary antibody for 1 h at room temperature. Quantification of bands was performed using ImageQuant software (Molecular Dynamics). Background intensity was subtracted from each sample and then fold change was determined. Fluorescent blots were imaged on the Odyssey Infrared Imaging System (LI-COR Biosciences). To verify equal loading, membranes were either cut in half and probed for target and loading controls simultaneously or stripped and re-probed for GAPDH as appropriate.
Ripa buffer
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Germany, Japan, Canada
RIPA buffer is a detergent-based buffer used for the extraction and solubilization of proteins from cells and tissues. It contains a combination of ionic and non-ionic detergents, as well as other components that help to disrupt cell membranes and solubilize proteins. The buffer is commonly used in protein analysis techniques such as Western blotting and immunoprecipitation.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (22 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (21 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (19 mentions)
Nitrocellulose membrane, by Bio-Rad (16 mentions)
Bca protein assay, by Thermo Fisher Scientific (13 mentions)
Pvdf membrane, by Bio-Rad (12 mentions)
β actin, by Merck Group (11 mentions)
Chemidoc xr, by Bio-Rad (11 mentions)
Protease inhibitor cocktail, by Roche (11 mentions)
β actin, by Santa Cruz Biotechnology (9 mentions)
Protein assay, by Bio-Rad (8 mentions)
Bca assay, by Thermo Fisher Scientific (7 mentions)
C digit blot scanner, by LI COR (7 mentions)
Bcl 2, by Santa Cruz Biotechnology (7 mentions)
Sds page, by Bio-Rad (7 mentions)
Quantity one software, by Bio-Rad (7 mentions)
β actin, by Cell Signaling Technology (7 mentions)
Benzonase, by Merck Group (7 mentions)
Bis tris gel, by Thermo Fisher Scientific (7 mentions)
Protease inhibitor cocktail, by Santa Cruz Biotechnology (6 mentions)
365 protocols using ripa buffer
1
Protein Extraction and Western Blot Analysis
2
Glucose and Lactate Measurement in NCI-737 Treated Cells
3
Protein Extraction and Quantification
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For protein extraction cells were sonicated in ice-cold 1X RIPA buffer (Santa Cruz Biotechnology) containing protease and phosphatase inhibitor (Roche Applied science, Indianapolis, IN). Lysates were incubated on ice for 30 min and centrifuged at 10,000 × g for 10 min at 4 °C. Supernatant fluid was collected and used as a total cell lysate for protein assays. Protein concentration was measured by BCA protein assay. Equal amounts of protein (50 μg) were used for ELISA.
4
Protein Quantification and Immunodetection
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The cell pellets were lysed using ice‐cold 1x RIPA buffer (Santa Cruz Biotechnology) followed by sonication, and the content was agitated for 30 mins on ice. Then, the tubes were centrifuged at 40C and the supernatant was collected in fresh tubes. Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher). The protein levels were assessed using automated protein separation and immunodetection, Jess Simple Western System, and all of the steps were performed using the instructions and reagents provided by the manufacturer (ProteinSimple). In brief, each sample was diluted to 0.5 ug/ul in 0.1x sample buffer, combined with 4x fluorescent master mix in 4:1 ratio and denatured at 950C for 5 mins accompanied by biotin ladder. The tubes were quickly vortexed and centrifuged for a min, and then, the recommended volume for samples, appropriate dilution of primary and secondary antibodies and luminol‐peroxide mix were loaded into a 12–230 kDa Jess 13‐well capillary plate for separation, centrifuged and then ran in the Jess instrument. The list of the primary and secondary antibodies is provided in Table S2 . The relative protein expression was determined by the ratio of area under the peak to that of βeta‐actin generated by the Compass for SW software 5.0.1 (ProteinSimple).
5
Silencing NAMPT Inhibits Proliferation
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Lipofectamine RNAiMAX (ThermoFisher) was used according to the manufacturer’s instructions to deliver 25 pM siNAMPT (Qiagen: Hs_PBEF1_1,3,5,6) to 250,000 cells/well in six-well plates, 24 h after seeding. Protein was harvested 72 h post-transfection in 1X RIPA buffer (Santa Cruz Biotechnology) for western blot. For proliferation assays, cells were plated at 10 000 cells/well in six-well plates and siRNA was delivered as described above when cells had reached 20% confluency for TC71/TC32, and 30% confluency for RDES. Proliferation assays were repeated at least three times. Between 4- and 6-days post-transfection, cells were stained with NeatStain (AstralDiagnostics, West Deptford, NJ) and images were acquired.
6
Fractionation of Detergent-Soluble and Resistant Cellular Compartments
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LNCaP, C4, C4-2 and C4-2B cells were cultured as described earlier in 60 mm plates and grown to 80% confluency. The cells were fractionated into detergent soluble and detergent resistant fractions as originally described by Hamaguchi and Hanafusa (28) . Briefly, the CSK buffer used consists of 10 mM Pipes pH 6.8, 100 mM KCl, 2.5 mM MgCl2, 1mM CaCl2, 0.3 M sucrose, 1mM phenylmethylsulfonyl fluoride (PMSF), 1% Trasylol (Aprotinin), 1mM sodium orthovanadate, 10µM sodium molybdate, 1% Triton X-100. Grown cells were washed with Tris-buffered saline and incubated with 0.5 ml of CSK buffer for 3 min on ice with gentle shaking every 30 sec. The supernatant was collected and the insoluble matrix structure that remained on the dish was again incubated for 1 min on ice with an additional 0.5 ml of fresh CSK buffer. The supernatants from the two incubations were pooled and used as the detergent soluble fraction. The residual structure remaining on the dish was collected with 0.3 ml of 1X RIPA buffer ( Santa Cruz Biotechnologies, Inc) supplemented with 2mM sodium ortho-vanadate and 1X protease inhibitor cocktail ( Santa Cruz) and used as the detergent resistant fraction.
Both fractions were centrifuged at 15,000g for 30 min and the supernatants were used for analysis.
Both fractions were centrifuged at 15,000g for 30 min and the supernatants were used for analysis.
7
Skin Tumor Induction and Protein Extraction
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All experiments involving mice were carried out in compliance with the Guide for the Care and Use of Laboratory Animals, and protocols approved by the Animal Care and Use Committee of the Pennsylvania State University College of Medicine. For mouse experiments, the dorsal surface of FVB/N mice (6-8 weeks) was treated with a single application of DMBA followed by multiple applications of TPA (25 weeks, 2X/week) to induce tumors, as described previously (Carr et al. 2012) (link). Animals were euthanized, and dorsal skin was treated with a depilatory agent for 3 min, washed, and tumors were removed. Tumor samples were placed into ice-cold 1X RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1 % NP-40, 1 % sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM-beta-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, 1 mM PMSF, and 1X protease inhibitor cocktail) (Santa Cruz Biotechnology, Santa Cruz, CA). Following sonication, samples were centrifuged, and the supernatants were collected. Samples were flash-frozen and later processed in RIPA buffer by homogenizing for 30 s on ice using a Polytron homogenizer, tissue was centrifuged at 30,000×g for 30 min at 4 °C, and supernatants were collected. Equal amounts of protein were then subjected to SDS-PAGE.
8
Western Blot Analysis of Protein Levels
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For protein level analyses, proteins were extracted from germ cells using RIPA buffer (Santa Cruz) containing 1× protease inhibitor cocktail (Roche). Protein concentration was calculated using a BCA protein assay kit (Pierce). Lanes of 4–15% gradient SDS polyacrylamide gels (Bio-Rad) were loaded with 20 µl of 1 mg/ml protein extract. Following protein separation via standard SDS PAGE, proteins were transferred to PVDF membranes using the Trans-Blot® Turbo™ western transfer system (Bio-Rad). Primary antibodies and dilution used are presented in Supplemental Table S2 . At a 1∶20,000 dilution, Invitrogen horseradish peroxidase-conjugated antibodies rabbit anti-mouse (R21455), goat anti-rabbit (A10533), rabbit anti-goat (R21459) were used as secondary antibodies. The presence of antibodies on the PVDF membranes was detected via treatment with Pierce ECL western blotting substrate (Thermo Scientific) and captured using the Syngene XR5 gel documentation system. Protein levels were assessed using Image J (NIH). The SMC3 Co-IP experiment was performed using the Dynabead® Co-IP kit (Life Technologies). Each milligram of beads was covalently linked to 4 µg of SMC3 antibody (Abcam, ab9263) or corresponding IgG control antibody (Life Technologies, A10533).
9
Protein Isolation and Western Blot Analysis
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For protein isolation, cells were lysed with a radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, Heidelberg, Germany) 72 h after transfection. After lysis, cells were centrifuged, and the clarified supernatant was used for analysis. Western blot analysis was performed as previously described [5 (link)]. Type VII collagen was detected using a polyclonal anti-collagen type VII rabbit antibody or human-specific anti-collagen type VII antibody specific for the NC1 and NC2 domains [59 (link)], and as a loading control, a β-tubulin-specific antibody (ab6064; Abcam, Cambridge, UK) was used. Goat anti-rabbit HRP-labelled antibody (Dako, Santa Clara, CA, USA) was used as a secondary antibody. Protein bands were visualized with the Immobilon Western Chemiluminescent HRP Substrate (Merck, Darmstadt, Germany) using the ChemiDoc XRS Imager (BioRad, Hercules, CA, USA). Native C7 Western blots were performed as previously described [60 (link)].
10
Western Blot Analysis of MMP3 and IRF8
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The indicated cell lines (3 × 106 cells) were plated overnight. Protein lysates were generated in RIPA buffer (Santa Cruz, Dallas, TX) supplemented with protease inhibitors (100X Halt protease cocktail mix, Thermo Fisher Scientific, Waltham, MA). Total protein was measured by the BCA assay (Thermo Fisher Scientific). Proteins were then separated by 10% gel electrophoresis, followed by transfer to nitrocellulose membrane (Bio-Rad, Hercules, CA). Membranes were incubated sequentially with mouse anti-MMP3 antibody (1:500 dilution; Abbiotech, San Diego, CA) or rabbit anti-IRF8 (Cell Signaling, Danvers, MA) and detected using HRP-conjugated secondary antibody (1:5000 dilution; Promgea, Madison, WI). Bands were visualized by exposure to Chemi-luminescent substrate (Thermo Fisher Scientific). The blot was then stripped and probed again for total actin using anti-mouse anti-actin antibody (1:2000 dilution; Promega).
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