Isolation of chromatin-associated RNA was performed as previously described with minor modifications (Conrad and Ørom, 2017 (link)). Approximately 5x10ˆ6 cells were washed with ice-cold PBS twice and scrapped. The cells were lysed with 400μl ice-cold lysis buffer (10 mM Tris-Hcl pH 7.5, 150 mM NaCl, 0.15% IGEPAL, 20U/μl SUPERSase-IN, 1x proteinase inhibitor Complete (Roche)) and incubated on ice for 5 min. After the incubation, 1 mL of ice-cold sucrose buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 24% Sucrose, 20U/μl SUPERSase-IN, 1x proteinase inhibitor Complete (Roche)) was under-laid and then the nuclei were collected under 16000 g centrifuge at 4C for 10 min. Isolated nuclei were resuspended in 250μl of ice-cold glycerol buffer (20 mM Tris-Hcl l pH 7.5, 75mM NaCl, 0.5 mM EDTA, 20U/μl SUPERSase-IN, 1x proteinase inhibitor Complete (Roche)) followed by 250μl of ice-cold nuclear lysis buffer (10 mM Tris-Hcl pH 7.5, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% IGEPAL, 20U/μl SUPERSase-IN, 1x proteinase inhibitor Complete (Roche)). Two minutes incubation was carried out on ice with mixing by max speed vortex for 5 s every minute and then chromatin pellets were precipitated under 13,000 g centrifuge at 4C for 2 min. Chromatin pellets were resuspended in 1ml of Trizol reagent, and RNA isolation was performed following manufacturer’s instructions.
Complete proteinase inhibitor
Manufactured by Roche
Sourced in United States, Switzerland
Complete proteinase inhibitor is a laboratory product that functions to inhibit a wide range of proteolytic enzymes, also known as proteinases or proteases. It is designed to prevent the degradation of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2 affinity gel, by Merck Group (5 mentions)
Anti cdc2 y100, by Abcam (5 mentions)
Anti myc resin 9e10, by Santa Cruz Biotechnology (4 mentions)
Anti ccq1 rabbit serum, by Abcam (3 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Anti myc 9e10, by Santa Cruz Biotechnology (2 mentions)
Glutathione sepharose 4b, by GE Healthcare (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Protein g agarose, by Roche (2 mentions)
Monoclonal anti ha f 7, by Santa Cruz Biotechnology (2 mentions)
Phosstop phosphatase inhibitor, by Roche (2 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions)
γ h2ax, by Abcam (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Heparinase 3, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
Hif 1α, by GeneTex (1 mentions)
Imagequant las 4000 detection system, by GE Healthcare (1 mentions)
54 protocols using complete proteinase inhibitor
1
Chromatin-associated RNA Isolation and Extraction
2
Protein Expression Analysis in Cell and Tissue Samples
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RIPA buffer was used to lyse cells (50 mM Tris-Cl, pH 7.5, 1% NP-40, 0.5% Na-deoxycholate, 150 mM NaCl supplemented with complete proteinase inhibitor, Roche Applied Sciences) and mice carotid arteries (50 mM Tris-Cl, pH 7.5, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM EDTA, 150 mM NaCl supplemented with complete proteinase inhibitor). Equal amounts of protein (30–60 μg) were separated by 10% SDS-PAGE, and electrotransfered to a PVDF membrane. Membranes were blocked with 5% milk in TBS for 1 h at room temperature, and incubated with primary antibodies against p53 (1:500, Abcam), p21 (1:200, Abcam), PCNA (1:1000, Santa Cruz), γ-H2AX (1:500, Abcam), GAPDH (1:1000, Cell Signaling Technology).
3
Heparinase Treatment of Cellular Lysates
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For heparinase treatment, cells were lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5% Triton X-100 plus complete proteinase inhibitor (EDTA free; Roche) on ice for 30 min. Supernatant was diluted with equal volumes of 20 mM Tris-HCl, pH 7.5, and 4 mM CaCl2 plus complete proteinase inhibitor (EDTA free; Roche). Heparinase III (Sigma-Aldrich) was added to a final concentration of 2 U/ml. After 4 h of incubation at 37°C, the reaction was halved and the two aliquots were mixed with NuPage LDS sample buffer (Invitrogen) with (for reducing gels) or without β-ME (for nonreducing gels) and run on 10% Mini-Protean TGX precast gels (Bio-Rad Laboratories).
4
Protein Expression Analysis of Cell and Tissue Lysates
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RIPA buffer was used to lyse cells (50 mM Tris-Cl, pH 7.5, 1% NP-40, 0.5% Na-deoxycholate, 150 mM NaCl supplemented with complete proteinase inhibitor, Roche Applied Sciences) and mice muscles (50 mM Tris-Cl, pH 7.5, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM EDTA, 150 mM NaCl supplemented with complete proteinase inhibitor). Equal amounts of protein (30~60 μg) were separated by 10% SDS-PAGE, and electrotransfered to a PVDF membrane. Membranes were blocked with 5% milk in TBS for 1 h at room temperature, and incubated with primary antibodies against HIF1α (GeneTex), VEGFA (Arigo), β-actin (Cell Signaling Technology), Lamin A/C (Cell Signaling Technology), Tubulin (Cell Signaling Technology), α-SMA, SM22α, MMP2, MMP9, OPN and NG2 at 4℃ overnight, and then with the HRP-conjugated secondary antibody (Abcam) for 1 h. The blots were evaluated with GE ImageQuant™ LAS 4000 detection system. The protein bands of interest were quantified using Image Pro Plus 6.0 software, and the integrated signal densities were normalized to β-actin or Tubulin (the loading control).
5
Protein Extraction from Mouse Lung
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E14.5 mouse lungs were dissected in cold PBS and minced finely using a razor blade. The tissue was then incubated with plain EBM (Lonza, Switzerland) or EBM containing 50 ng/ml VEGF for 15 min at 37°C. Lysis buffer (50 mM Tris/HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and 2 mM DTT) containing complete proteinase inhibitors (Roche, Switzerland), PhosSTOP (Roche, Switzerland), and sodium orthovanadate was added to the tissue, which was then pulverized with a pestle and incubated for 30 min while rotating at 4°C. Tissue was spun down and protein quantification was performed. The tissue was treated as described in the Western blotting section.
6
VEGFR2 Immunoprecipitation in HEK293T Cells
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HEK293T cells were transfected with the indicated constructs using Lipofectamine-2000 (Invitrogen, Carlsbad, CA). They were then grown in DMEM + 10% fetal bovine serum + 1% Penicillin-Streptomycin and 48 hr after transfection cells were washed and harvested in ice-cold PBS. Cells were lysed using lysis buffer (50 mM Tris/HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and 2 mM DTT) containing complete proteinase inhibitors (Roche, Switzerland). After 30 min of rotation in the cold room and subsequent centrifugation, protein was quantified and 20 µg of protein was frozen down as input controls. 0.5 µg of anti-VEGFR2 antibody (gift of Procter and Gamble, see Gu et al., 2003 (link) for details) was added to 500 µg of protein and rotated in the cold room for 1 hr. Then, 20 µl of protein A/G beads (Thermo Scientific, Waltham, MA) were added to the protein and rotated overnight in the cold room. Beads were washed 3× with lysis buffer and two times with wash buffer (lysis buffer with 300 mM NaCl). Protein was eluted by the addition of 2× SDS-PAGE sample buffer and boiling for 10 min. Co-immunoprecipitation was also performed on P7 lung lysates isolated from control and Nrp1VEGF− animals treated with VEGF as described above.
7
Western Blot Analysis of PD-L1 Expression
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Cultured cells were homogenized in lysis buffer containing 50 mmol/L Tris‐HCl, pH 7.4, 150 mmol/L NaCl, 10 mmol/L CaCl2, 1% NP‐40, complete proteinase inhibitors (Roche Diagnostics) and homogenate supernatants were used for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.18 Proteins were transferred to polyvinylidene difluoride membranes, and the membranes were incubated with rabbit anti–PD‐L1 antibody (E1L3N; Cell Signaling Technology) or rabbit anti–β‐actin antibody conjugated with HRP (13E5; Cell Signaling Technology). For anti–PD‐L1 antibody, the membranes were subsequently incubated with secondary antibodies. They were further visualized by chemiluminescence.
8
Tissue Lysis and Immunoblot Analysis
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The brain and kidney were isolated and lysed in an extraction buffer containing 50 mM Tris-HCl (pH7.4), 1% sodium dodecyl sulphate (SDS) plus Complete proteinase inhibitors (Roche) and 1 mM phenylmethylsulfonyl fluoride. Protein concentration in the lysate was determined by the bicinchoninic acid colorimetric assay (Pierce). For the immunoblot analysis, 10 μg protein was loaded onto 5% to 20% SDS-polyacrylamide gel, electrophoresed, and immunoblotted as previously described19 (link). Subunit G isoforms were detected by isoform-specific antibodies using the Pierce Western Blotting Substrate Plus system. Images were acquired and analyzed using the GE Healthcare LAS-4000 mini system37 (link).
9
Crosslinking and Chromatin Extraction in Mouse ESCs
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Chromatin was extracted and fixed as described previously104 (link). In brief, 10 × 106 mouse ESCs were trypsinized, collected in 50 ml falcon tubes in 9.3 ml medium, and crosslinked with 1.25 ml 16% formaldehyde (1.89% final concentration; methanol-free, Thermo Fisher Scientific) while rotating for 10 min at 25 °C. Cells were quenched with 1.5 ml 1 M cold glycine, washed with cold PBS and lysed for 20 min at 4 °C in lysis buffer (10 mM Tris pH 8, 10 mM NaCl, 0.2% NP-40, supplemented with cOmplete proteinase inhibitors (Roche)) prior to snap freezing in 1 ml lysis buffer on dry ice. Fixed chromatin was stored at −80 °C.
10
Synovial Tissue Protein Extraction
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Synovial tissue protein was extracted using PhosphoSafe buffer (Novagene, Madison, WI) supplemented with Complete Proteinase Inhibitors (Roche Applied Science, Indianapolis, IN, USA). RIPA buffer was used for protein extraction from synovial tissue. The protein concentrations of tissue and FLS were determined using the Micro BCA protein assay kit (Thermo Scientific, Rockford, IL). Samples containing 25 μg of protein from cultured FLS or 50 μg of protein from synovial tissue were resolved on Invitrogen (Carlsbad, CA, USA) NuPage 4 to 12% precast gels and transferred to a PVDF membrane. Membranes were developed with Immun-Star Western ECL substrate (Bio-Rad, Hercules, CA, USA) and imaged on VersaDoc imaging system (Bio-Rad), using QuantityOne software (Hercules, CA, USA) for image capture and densitometry.
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