Western blot analysis was performed according to a previous study (29 (link)). Briefly, tissue samples were homogenized in a RIPA buffer (Qiagen, Inc.) supplemented with a cocktail of proteinase inhibitors and with a cocktail of phosphatase inhibitors (both Roche Diagnostics). Protein concentrations were determined using a bicinchoninic acid kit (Pierce; Thermo Fisher Scientific, Inc.). Proteins (20 µg/lane) were separated using 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% bovine serum albumin/PBS at room temperature for 1 h, the membranes were incubated with primary LBP antibody (anti-LBP antibody; 1:1,000 dilution; cat. no. ab169776; Abcam) and β-actin (1:1,000 dilution; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C. After washing with Tween-20 (0.05%) in PBS, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:2,000 dilution; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. LBP and β-actin were detected using ECL development solution (Pierce; Thermo Fisher Scientific, Inc.). LBP and β-actin expression levels were determined using Quantity One v4.6.2 software (Bio-Rad Laboratories, Inc.). β-actin was used as a loading control.
Ripa buffer
Manufactured by Qiagen
Sourced in China
RIPA buffer is a protein extraction and lysis reagent used in biological research. It is a mixture of detergents, salts, and buffers that help solubilize and extract proteins from cells and tissues. RIPA buffer is commonly used to prepare cell and tissue lysates for various downstream applications, such as protein quantification, Western blotting, and immunoprecipitation.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Cocktail of phosphatase inhibitors, by Roche (3 mentions)
Proteinase inhibitor, by Roche (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
β actin, by Abcam (1 mentions)
β actin, by Bio-Rad (1 mentions)
Quantity one v4, by Bio-Rad (1 mentions)
Bca assay, by Bio-Rad (1 mentions)
Cocktail of proteinase inhibitors, by Roche (1 mentions)
Anti tgf β1, by Abcam (1 mentions)
Anti β tubulin antibody, by Abcam (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Lumi film chemiluminescent detection film, by Roche (1 mentions)
Horseradish peroxidase labeled secondary goat anti mouse igg antibody, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Cd54 icam 1, by Cell Signaling Technology (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
12 protocols using ripa buffer
1
Quantitative Analysis of LBP Protein Expression
2
Evaluating c-Met Signaling in Breast Cancer Xenografts
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The PD studies were conducted in athymic nude mice bearing MDA-MB-231 tumor xenografts by determining the effects of HVS on c-Met signaling. Mice were humanely euthanized at designated times following HVS administration, and resected breast tumor tissues were stored at −80°C until protein extracted. Breast tumor tissues were homogenized in RIPA buffer (Qiagen Sciences Inc., CA) using an electric homogenizer. Protein lysates were generated, and protein concentrations were determined using the BCA assay (Bio-Rad Laboratories, CA). The levels of total and phosphorylated c-Met were determined using Western blot analysis. The visualization of β-tubulin was used to ensure equal sample loading in each lane. The experiment was repeated three times and representative Western blot images are shown in Figure 10D .
3
Western Blot Analysis of NPC2 and NPC1L1
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Western blot was performed according to previous study 15 . Briefly, tissue samples were homogenated in a RIPA buffer (Qiagen, China) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science). The protein concentrations were determined using the Bicinchoninic Acid (BCA) Kit (Pierce). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were incubated with primary NPC2 antibodies (A5413; Abclonal, USA) at 1:1500, or NPC1L1-HRP (ab201773; Abcam, USA): 1:2000) overnight at 4 °C. After washing the membrane, NPC1L1 was visualised using ECL development solution. For detection of NPC2, secondary antibodies incubated at room temperature for 1~2 h and NPC2 was visualised using ECL development solution.
4
Liver Protein Expression Analysis
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The liver tissues were lysed at 4°C in RIPA buffer (Qiagen) and extracts clarified at 12,000×g for 25 min. Aliquots (60–90 µg protein) were electrophoretically separated, transferred to nitrocellulose membranes, incubated overnight with specific primary antibodies (anti-TGF-β1, 1 : 2000, anti-α smooth muscle actin (anti-α-SMA) antibody, 1 : 1000; anti-CCN4 antibody, 1 : 500; 1 : 1000; anti-β-tubulin antibody, 1 : 5000; all from Abcam). After washing three times, primary antibodies were detected with secondary antibody (Abcam, 1 : 5000). Immunoreactive proteins were visualized with ECL reagent and quantitated by densitometry. Activity of nuclear factor-κB (NF-κB) was determined by active NF-κBp65 (Abcam, 1 : 2000) using Western blot. Histone H3 was used as a nuclear internal control.
5
Protein Expression Analysis of Colon Tissue
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Frozen colon tissue samples were lysed in RIPA buffer (Qiagen) followed by centrifugation (12,000 rpm, 4°C, 15 minutes), after which the supernatants were stored at −80°C until use. Extracted proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membrane was blocked with Tris-buffered saline containing 0.1% Tween 20 (pH 7.6) for 1 hour at room temperature. Subsequently, the PVDF membrane was immunoblotted overnight at 4°C with the primary antibody solution (1 : 1,000). After washing twice with TBST, the membrane was incubated with horseradish peroxidase-labeled secondary goat anti-mouse IgG antibody (Santa Cruz Biotechnology) for 1 hour at room temperature and thereafter washed three times with TBST. The final detection was performed with enhanced chemiluminescence Western blotting reagents (GE Healthcare Bio-Sciences Corp.) and the membranes were exposed to Lumi-Film Chemiluminescent Detection Film (Roche Applied Science).
Loading differences were normalized by using the housekeeping control GAPDH. The primary antibodies used in this study included anti-omentin-1 and anti-GAPDH (both from Santa Cruz Biotechnology).
Loading differences were normalized by using the housekeeping control GAPDH. The primary antibodies used in this study included anti-omentin-1 and anti-GAPDH (both from Santa Cruz Biotechnology).
6
Quantifying Autophagy and Apoptosis Markers
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Tissue and cell samples were homogenized in a RIPA buffer (Qiagen, China) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and phosphatase inhibitors (Roche Applied Science, Switzerland). Protein concentrations were determined using the Bicinchoninic Acid (BCA) Kit (Pierce). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), which were incubated with primary antibodies for LC3-I/II (CST, #4108, 1:1000), p62 (CST, #88588, 1:1000), p-AMPKα1/2 (R&D, AF2509, 1:2000), t-AMPKα (CST, #5831,1:1000), p-mTOR (CST, #5536, 1:1000), t-mTOR (CST, #2972, 1:1000), cleaved caspase-3 (CST, #9664, 1:1000), PARP (CST, #9532, 1:1000), Bcl-xl (CST, #2764, 1:1000), Mcl-1 (CST, #94296, 1:1000), or β-Actin (Sigma, A2066, 1:2000) overnight at 4 °C and probed with secondary antibodies at room temperature for 1~2 h. After washing the membranes, an enhanced chemiluminescence detection (ECL) reagent was used to expose the protein bands in a digital chemiluminescence imaging system (Bio-Rad).
7
Western Blot Analysis of Tumor Tissue Protein Markers
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Cells were homogenized and lysed in radioimmunoprecipitation assay (RIPA) buffer (Qiagen Sciences Inc., Valencia, CA, USA) and cleared by centrifugation. Animal tumor tissues were homogenized with a 1× protease inhibitor and cleared by centrifugation. Samples were resolved on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride membranes, and Western blotting analysis was performed as previously described [20 (link),36 (link),42 (link),43 (link)]. Actin presented in the images represents the loading protein control for one or more markers from the same cell lysates. Chemiluminescence was used to detect the positive bands on the membrane. [20 (link),36 (link),42 (link),43 (link)].
8
Inflammatory Protein Regulation by Eicosanoids
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HRMEC were seeded in 10 cm2 dishes and were placed in serum-reduced medium at 85% confluence. Cells were then treated with vehicle or 1 ng/ml TNFα in the presence or absence of 11,12-EET (0.5 μM) or 19,20-EDP (0.5 μM) with AUDA (10 μM) for 4 hours. Cells were then washed twice in cold PBS and lysed using radio-immunoprecipitation assay (RIPA) buffer (Qiagen) with protease inhibitors (Roche; Basel, Switzerland). Samples were equilibrated for total protein concentration using a Pierce BCA assay, subjected to 10% SDS-PAGE, and gels were transferred to nitrocellulose membranes using the iBlot system (Thermo Fisher Scientific). Membranes were blocked and probed in 5% BSA for CD54/ICAM-1 (1:1000; Cell Signaling) and β-Actin (1:4000; Thermo Fisher Scientific) or 5% milk for VCAM-1 (1:1000; Abcam; Cambrdige, UK). Blots were then labeled with horseradish peroxidase-conjugated secondary antibodies (1:2000). β-Actin was used as a loading control. Membranes were incubated in Pierce ECL Western blotting substrate and developed with a ChemiDoc MP (Bio-Rad; Hercules, CA). Blots were then quantified using ImageJ software.
9
Overexpression of Human SPARC in RAW 264.7 Cells
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The RAW 264.7 cell line was purchased from ATCC (ATCC TIB-71). The cells were cultured in DMEM with high glucose (Thermo Fischer Scientific) including 10% FBS (Omega Scientific) and 1% antibiotics/antimycotic (Thermo Fischer Scientific) and plated in 24-well plates as 1 × 106 cells per well. The plated cells were transfected with a vector containing human SPARC ORF or a mock vector (GeneCopoeia) using Lipofectamine 3000 (Thermo Fisher Scientific) following manufacturer’s instruction. After 4 hours of transfection, the media was replaced and cells were collected with RIPA buffer or RLT buffer (Qiagen) for Western blot or quantitative-PCR (qPCR) analysis after 48 hours of transfection.
10
Western Blot Analysis of KMO Protein Expression
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Briefly, tissue and cell samples were homogenated in a RIPA buffer (Qiagen,
China). After centrifugation at 12,000 g, 4 °C for
30 min, 50 μg of protein samples were fractionated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to nitrocellulose membranes. After blocking non-specific binding
sites for 60 min with 5 % non-fat milk, the membranes were incubated
with rabbit monoclonal antibody against KMO (1:1,800; LifeSpan BioSciences,
Inc.), or β-actin (1:1,000;Santa Cruz) at 4 °C
overnight, respectively, and subsequently, probed with HRP-conjugated
anti-rabbit secondary antibody (1:5,000; Santa Cruz) for 45 min at room
temperature. Chemiluminescence detection was performed using SuperSignal West
Femto Maximum Sensitivity Substrate Kit 19 (Pierce). Membranes were exposed and
recorded with Molecular Imager ChemiDoc XRS+System (Bio-Rad, CA, USA).
China). After centrifugation at 12,000 g, 4 °C for
30 min, 50 μg of protein samples were fractionated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to nitrocellulose membranes. After blocking non-specific binding
sites for 60 min with 5 % non-fat milk, the membranes were incubated
with rabbit monoclonal antibody against KMO (1:1,800; LifeSpan BioSciences,
Inc.), or β-actin (1:1,000;Santa Cruz) at 4 °C
overnight, respectively, and subsequently, probed with HRP-conjugated
anti-rabbit secondary antibody (1:5,000; Santa Cruz) for 45 min at room
temperature. Chemiluminescence detection was performed using SuperSignal West
Femto Maximum Sensitivity Substrate Kit 19 (Pierce). Membranes were exposed and
recorded with Molecular Imager ChemiDoc XRS+System (Bio-Rad, CA, USA).
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