To analyze protein expression in the mouse small intestine, the tissue was minced with cold radioimmunoprecipitation assay buffer (RIPA) buffer (Rockland, Limerick, PA, USA), supplemented with 1 mM dithiothteitol (DTT) (Merk, Darmstadt, Germany), and diluted with a phosphatase inhibitor cocktail 2 solution (Merk). After centrifugation, the amount of protein in each supernatant was quantified, mixed with an SDS sample buffer, and denatured for 5 min at 95 °C. Protein electrophoresis and transfer as well as membrane development were all performed as described previously [23 (link),24 ]. Briefly, electrophoresis was performed using 12% Tris-glycine SDS-polyacrylamide gel, and the protein bands were transferred onto a polyvinylidene difluoride membrane, which was blocked for unspecific binding of proteins by incubation at room temperature with 5% skim milk. After three washes with a Tris-Buffered Saline buffer containing 0.1% Tween® 20, the membrane was incubated with anti-α-defensin-1 or β-Actin antibodies for 3 h, followed by washes and incubation with a secondary antibody. The proteins were detected by using the FUSION Solo Vilber Lourmat system (Collégien, France) with an ECL solution. The intensities of the protein bands were quantified using the ImageJ program and captured images.
Ripa buffer
Manufactured by Rockland Immunochemicals
Sourced in United States
RIPA buffer is a detergent-based buffer solution used for cell lysis and protein extraction. It contains a combination of ionic and nonionic detergents, as well as other components to solubilize and extract proteins from cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Dithiothreitol (dtt), by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Prednisolone, by Merck Group (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid bca reagent, by Thermo Fisher Scientific (1 mentions)
Ecl detection reagent, by GE Healthcare (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Muse annexin 5 dead cell kit, by Merck Group (1 mentions)
Odyssey classic scanner, by LI COR (1 mentions)
Irdye 800cw donkey anti mouse igg, by LI COR (1 mentions)
Odyssey 9120, by LI COR (1 mentions)
Image studio software, by LI COR (1 mentions)
12 protocols using ripa buffer
1
Protein Expression Analysis in Mouse Intestine
2
Protein Expression Analysis in HFDPCs
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HFDPCs were treated with the indicated concentrations of CMX for 24 h. Following treatment, the cells were washed with phosphate-buffered saline (PBS) twice and then lysed in a cold radioimmunoprecipitation assay (RIPA) buffer (Rockland, Limerick, PA, USA) containing 1 mM DTT (Sigma, Saint Louis, MO, USA), a phosphatase inhibitor cocktail (Merck, Darmstadt, Germany), and a protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN, USA) [15 ]. The protein concentration of each sample was adjusted to a constant value after measurements using the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Next, cell lysates were resolved on an 8−10% sodium dodecyl sulfate-polyacrylamide gel and proteins were subsequently transferred to an Immobilon-P PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk prepared in Tris-buffered saline (TBS, pH 7.4) containing 0.1% Tween 20 (TBS-T) for 120 min and probed with specific primary antibodies overnight at 4 °C. The membrane was then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h and washed with TBS-T 3 times before visualization using an ECL system.
3
Cell Apoptosis Induction Analysis
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Huh7 and HepG2 cells were acquired from the American Type Culture Collection. The cells were maintained in DMEM containing 10% fetal bovine serum and 1% streptomycin/penicillin (all from Thermo Fisher Scientific, Inc.) at 37°C in a humidified 5% CO2 incubator. Prednisolone, MTT and doxorubicin were obtained from Sigma-Aldrich (Merck KGaA). The Muse® Annexin V Dead cell kit and cell cycle arrest package for flow cytometry analysis, and the PVDF membranes were purchased from EMD Millipore. For nick-end labeling, the DeadEnd™ Fluorometric TUNEL System was purchased from Promega Corporation. The human apoptosis proteome array kit was obtained from Bio-Rad Laboratories, Inc. The ECL detection reagent was bought from GE Healthcare. RIPA buffer was procured from Rockland Immunochemicals, Inc. Bicinchoninic acid (BCA) reagent was obtained from Thermo Fisher Scientific, Inc. The cytochrome c release kit (cat. no. ab65311) was from Abcam. Anti-GAPDH, anti-cleaved caspase 3, anti-cleaved caspase 9, anti-caspase 9, anti-cleaved poly-ADP ribose polymerase (PARP), anti-PARP, anti-caspase 3, anti-Bcl-2, anti-Bax, and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse (cat. nos. 2118, 9664, 9505, 9502, 9541, 9532, 9662, 2870, 5023, 7074 and 7076, respectively) were obtained from Cell Signaling Technology, Inc.
4
Western Blot Analysis of Endothelial Proteins
5
Western Blot Protein Analysis Protocol
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Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Rockland Immunochemicals, Pottstown, PA) supplemented with 1× Halt Protease Inhibitor Cocktail and centrifuged at 4°C for 5 min, 14,000g. Supernatants were collected and transferred to new tubes, and protein concentrations were quantified using the Pierce BCA Protein Assay Kit. For each sample, ~20 μg of protein was diluted to equal volume with water, mixed with sample buffer, and heated at 95°C for 5 min. Samples were resolved on 4 to 12% tris-glycine gels and transferred to nitrocellulose membranes. Membranes were blocked for 1 hour in tris-buffered saline plus 0.1% Tween 20 (TBST) supplemented with 5% nonfat dry milk. Primary antibodies (table S2) were diluted in TBST supplemented with 5% nonfat dry milk and were added to membranes and incubated overnight at 4°C on a rocking platform. Membranes were washed five times with TBST. Secondary antibodies (table S2) were diluted in TBST supplemented with 5% nonfat dry milk and were added to membranes and incubated for 1 hour at room temperature on a rocking platform, protected from light. Membranes were washed five times with TBST and imaged using an Odyssey 9120 (LI-COR, Lincoln, NE). Band intensities were quantified using Image Studio software (LI-COR).
6
LNCaP Cell Authentication and Western Blot Analysis
7
Protein Expression Analysis in Cisplatin-Treated Cells
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Cisplatin-treated and untreated cells were suspended in RIPA buffer (Rockland, Gilbertsville, PA, USA) containing 5 µM of AEBSF, 1.5 nM aprotinin 10 nM E-64, 10 nM leupeptin, sodium orthovanadate, sodium molybdate, sodium tartrate and imidazole, and were placed on ice for 20 minutes. The supernatant was collected after centrifugation at 4℃ at 13,000 rpm for 20 minutes. The protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). The whole lysate (20 µg) was resolved on 10% or 12.5% SDS-PAGE gel, transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA) by electroblotting and probed with human anti-β actin, anti-p53, anti-p21, anti-caspase 3, anti-caspase 7, anti-caspase 9, or anti-PARP. The membrane was washed with PBS-T (1×PBS and 0.05% Tween-20) and incubated for 1 hour with an HRP-conjugated anti-rabbit or anti-mouse antibody. The blot was developed using an enhanced chemiluminescence kit (iNtRON Biotech, Seongnam, Korea).
8
Vitreous Humor VEGF Quantification
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Vitreous samples were collected using glass micropipettes. A micropipette puller is used to pull the tips of standard glass pipettes. Mice were anesthetized and the tip was inserted through the sclera 2 mm posterior to the limbus. Suction was activated by stepping on the foot pedal with a pump in reverse mode and vitreous was slowly aspirated, allowing collection of as much as 4 μL. Samples were immediately placed on ice, and the samples were centrifuged at 6000 rpm for 30 sec19 (link). 4 μL vitreous humor (from a single eye) was diluted in 6 μL RIPA buffer (Rockland Immunochemicals, PA, USA) containing protease inhibitor (Quartett, Berlin, Germany). VEGF protein levels of the vitreous humor samples were measured using a mouse VEGF Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.
9
Western Blot Analysis of HUVECs Treated with PLE-II
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HUVECs were treated with PLE-II at the indicated concentrations for 24 h. After treatment, the cells were washed with PBS and lysed in cold RIPA buffer (Rockland, Limerick, PA, USA) supplemented with 1 mM DTT (Merck, Darmstadt, Germany) and diluted protease inhibitor cocktail tablets (Sigma, St. Louis, MO, USA). After centrifugation, the amount of protein in each supernatant was quantified, mixed with SDS-sample buffer, and denatured for 5 min at 95 °C. Electrophoresis was performed using 10–12% tris-glycine SDS-polyacrylamide gel. Protein bands were transferred to polyvinylidene fluoride membranes, which were blocked at room temperature with 5% skim milk. After three washes with tris-buffered saline containing 0.1% Tween® 20, the membranes were incubated with a specific antibody for 3 h, followed by washes and incubation with a secondary antibody. The protein bands were visualized with the SuperSignalTM West Pico PLUS Chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA) using the Fusion Solo System (Vilber Lourmat, Paris, France).
10
Protein Extraction and Western Blot Analysis
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Cell lysates were prepared using RIPA buffer (Rockland Immunochemicals, PA, US; Cat.
No. MB-030-0050). Samples were centrifuged at 12,000 × g for 10 min at 4°C and the supernatant was isolated and quantified using a Pierce BCA protein assay kit (Thermo Fisher Scientific, MA, US). Loading buffer (4×) was added into the samples, and they were boiled for 5 min at 90°C. The extracted protein samples were separated on an 10% SDS-PAGE gel by electrophoresis and transferred to polyvinylidene fluoride membranes. Membranes were blocked in TBS-T (1% Tween 20) with 5% BSA for 30 min and incubated with primary antibody against human ABCD1 (1:1,000; Origene, MD, US; Cat. No. TA803208) and βactin (1:1,000; Santacruz, TX, US; Cat. No. sc-47778) for 40 h at 4°C. After washing, membranes were incubated with anti-mouse antibody (1:5,000; Merck Millipore, Germany; Cat. No. AP124P). After incubation, blots were visualized using the ECL western blotting detection reagent (GE Healthcare, IL, US; Cat. No. RPN2106) on an electrochemiluminescence 10 instrument (Amersham Biosciences, UK; Cat. No. RPN210).
No. MB-030-0050). Samples were centrifuged at 12,000 × g for 10 min at 4°C and the supernatant was isolated and quantified using a Pierce BCA protein assay kit (Thermo Fisher Scientific, MA, US). Loading buffer (4×) was added into the samples, and they were boiled for 5 min at 90°C. The extracted protein samples were separated on an 10% SDS-PAGE gel by electrophoresis and transferred to polyvinylidene fluoride membranes. Membranes were blocked in TBS-T (1% Tween 20) with 5% BSA for 30 min and incubated with primary antibody against human ABCD1 (1:1,000; Origene, MD, US; Cat. No. TA803208) and βactin (1:1,000; Santacruz, TX, US; Cat. No. sc-47778) for 40 h at 4°C. After washing, membranes were incubated with anti-mouse antibody (1:5,000; Merck Millipore, Germany; Cat. No. AP124P). After incubation, blots were visualized using the ECL western blotting detection reagent (GE Healthcare, IL, US; Cat. No. RPN2106) on an electrochemiluminescence 10 instrument (Amersham Biosciences, UK; Cat. No. RPN210).
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