Proteins were extracted from the NSCLC cells (n = 3, biological replicates) or tumor tissues in the lysis buffer (KeyGEN BioTECH, Nanjing, China) and protease inhibitor cocktail (KeyGEN BioTECH, Nanjing, China) for western blotting34 (link). Antibodies used for western blotting were: anti-IRX4 (ab123542, Abcam, Cambridge, UK), anti-pEGFR, EGFR, p-smad3, smad2/3 (#3777, #4267, #9520, #8685, Cell Signaling Technology, Boston, MA, USA), anti-CD133, NANOG, Notch1, β-catenin, MDR1, VDR, ABCG2 and anti-Histone-H3 (18470-1-AP, 14295-1-AP, 20687-1-AP, 51067-2-AP, 22336-1-AP, 14526-1-AP, 27286-1-AP, 17168-1-AP, Proteintech group, WUHAN SANYING, WuHan, China), anti-Sox2, Oct4, ALDH1A1 (D164316, D121072, D220058, Sangon Biotech, Shanghai, China), and anti-β-actin, anti-GAPDH (bs-0061R, bs-0755R, Bioss, Beijing, China) antibodies, HRP-conjugated goat anti-rabbit IgG secondary antibody (ABL3012-2, AbSci, Vancouver, WA, USA). The electrochemical luminescent substrates (Tanon, Shanghai, China) were used according to the manufacturer’s protocol in order to visualize the proteins of interesting in the Tanon imaging system. The relative expressions were quantified densitometrically using the ChemiScope analysis software and calculated according to the reference bands of GAPDH or β-actin.
Protease inhibitor cocktail
Manufactured by Keygen Biotech
Sourced in China, United States
The Protease inhibitor cocktail is a laboratory reagent designed to inhibit the activity of proteolytic enzymes, known as proteases. It is commonly used in biological research and analytical applications to prevent the degradation of proteins and peptides during sample preparation and processing.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Ripa buffer, by Keygen Biotech (4 mentions)
Bca protein assay kit, by Keygen Biotech (3 mentions)
Phosphatase inhibitor cocktail, by Keygen Biotech (3 mentions)
Lysis buffer, by Keygen Biotech (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ripa lysis buffer, by Keygen Biotech (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Collagen 1, by Abcam (2 mentions)
β actin, by Proteintech (2 mentions)
100 mm cell nylon mesh, by BD (2 mentions)
Endotoxin free collagenase 1, by Merck Group (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Anti p egfr, by Cell Signaling Technology (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
P smad3, by Cell Signaling Technology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
31 protocols using protease inhibitor cocktail
1
Protein extraction and western blotting
2
Quantification of Hippocampal Amyloid-Beta
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The hippocampus tissue was homogenized in RIPA buffer (Beyotime Biotechnology) containing a protease inhibitor cocktail (KeyGENBioTECH, catalog #KGP603), ultrasonicated, and centrifuged at 13000g for 10 min. The supernatant was collected and total protein was measured with a Micro BCA protein assay (Pierce). Aβ40 and Aβ42 were separately measured with ELISA Kits (R D system) by following manufacturer's instructions.
3
Cytoprotective Effects of FMN, APAP on Cells
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Dulbecco's modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco, Grand Island, NY, USA. The FMN, APAP and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Aldrich, Saint Louis, MO, USA. TdT-mediated biotin-dUTP nick-endlabeling (TUNEL) kit was purchased from Roche Diagnostics Gmbh, Mannheim, Germany. The kits for determining GSH and MDA contents were obtained from Jianchen, Nanjing, China. The kits for determining serum AST and ALT activity were obtained from Jianchen, Nanjing, China. Antibodies against Nrf2 (Cat# ab31163) were purchased from Abcam, Cambridge, MA and anti-β-actin (Cat#A1978) antibodies were from Sigma Aldrich, Saint Louis, MO, USA. BCA protein assay kit and enhanced chemiluminescent substrate kit were obtained from Pierce, Thermo Scientific, Rockford, IL, U.S.A. Protease inhibitor cocktail was purchased from KeyGen, Shanghai, China. Other reagents were of analytical grade.
4
Western Blot Analysis of Protein Expression
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The cells were seeded in 10×10 mm2 dishes. After treatment, cells were washed twice with cold phosphate buffer solution (PBS, Hyclone, USA) and then scraped off in 1000 μl lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF) and 0.1 % protease inhibitor cocktail (KeyGen, Nanjing, China) and centrifuged at 14,000 g at 4°C for 15 min. The supernatants were obtained as total proteins and stored at −80°C for further studies. The total proteins were electrophoresed by 10-12% SDS-PAGE gel, and transferred to polyvinylidene fluoride (PVDF, Millipore, USA) membranes, which was activated in methanol. The blots were probed or reprobed with antibodies. The membranes were probed using Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) and autoradiographed. The intensity of the bands was determined using densitometric analysis. The primary antibodies used were anti-rabbit RBM38, the alia name of RNPC1, (Santa Cruz, USA), ERα (Cell Signaling technology, USA), ERβ (Cell Applications, USA), anti-mouse Actin (Cell Signaling technology, USA). The anti-rabbit and anti-mouse secondary antibodies were from Cell Signaling technology. Actin was used to normalize protein loading. The antibodies were diluted according to the manufacturer's instructions.
5
Subcellular Protein Fractionation Protocol
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The cells were collected, washed twice with PBS and lysed with RIPA buffer (KeyGEN Biotech, China) added with protease inhibitor cocktail (KeyGEN Biotech, China). The lysates were centrifuged at 12,000 ×g for 10 min at 4 ℃ and the supernatant was collected as the total protein. The mitochondrial, nuclear and cytoplasmic proteins were prepared strictly according to the Cell Mitochondria Isolation Kit (Beyotime Biotechnology, China) and the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, China) with about 107 cells. The protein concentration was determined with a BCA assay (Generay Biotech, China).
6
AGO2-Bound RNA Isolation and Analysis
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The cells were subjected to washing steps twice with PBS and were lysed using the RIPA lysis buffer (Thermo Fisher) containing a protease inhibitor cocktail (Keygen). The lysate was subsequently incubated with magnetic beads conjugated with anti-AGO2 or the rabbit IgG antibody control. The magnetic beads were washed twice with high-salinity wash buffer (700 mM NaCl). The immunoprecipitated RNA was separated using the TRIzol reagent and analyzed via qRT-PCR.
7
Dihydroartemisinin Cytotoxicity Assay Protocol
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RPMI-1640 (Cat# 11875093) and fetal bovine serum (Cat# 10099-141) were purchased from Gibco (Grand Island, NY, USA). Dihydroartemisinin (DHA) was purchased from Sigma Aldrich (Cat# D7439, St. Louis, MO, USA). Antibodies against KDM3A (Cat# ab106456) was purchased from Abcam (Cambridge, London, UK), anti-p21 (Cat# sc-471) , anti-GAPDH (Cat# sc-25778) and anti-β-Actin (Cat# sc-8432) antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Protease inhibitor cocktail was purchased from KeyGen (Cat# KGP603, Jiangsu, China). BCA protein assay kit and enhanced chemiluminescent substrate kit were obtained from Pierce (Cat# 23225, Thermo Scientific, Rockford, IL, U.S.A). Cell Counting Kit-8 (CCK-8) was purchased from Med Chem Express (Cat# HY-K0301, Shanghai, China).
8
Quantitative Protein Expression Analysis
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Total cellular protein was extracted from cultured cell populations using the RIPA buffer (Thermo Scientific, USA) supplemented with a protease inhibitor cocktail (Keygen Biotechnology, China), following the manufacturer's instructions. The protein concentration was determined using the BCA Protein Assay Kit (Beyotime, China). Protein samples were loaded at a concentration of 10 μg per well on a 10 % dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (10 %, 15 wells, GenScript, China) and subsequently transferred to polyacrylamide fluoride (PVDF) membranes (Merck, China). The PVDF membranes were then blocked using 10 % skimmed milk powder in TBST (TBS with 0.1 % Tween) for 2 h. The primary antibodies used in this study were DSPP and DMP1 (Affinity, China), which were applied at a dilution of 1:3000. GAPDH (1:5000, Affinity, China) was utilized as the internal reference. Immune complexes were detected using an anti-rabbit secondary antibody (Abcam, USA). The membranes were washed with TBST four times for 10 min each, followed by incubation with anti-rabbit secondary antibodies for 2 h at room temperature. The Immunoreactive bands were then visualized using a chemiluminescence reagent (Merck Millipore, Darmstadt, Germany) and an imaging system (LAS4000 M). Densitometry analysis was performed using ImageJ software to quantify the observed bands.
9
Western Blot Analysis of Apoptosis-Related Proteins
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Heart or cell samples were lysed in RIPA buffer (KeyGEN, Nanjing, China) with protease inhibitor cocktail (KeyGEN) and concentration of protein sample was quantified by BCA Protein Assay Kit (TaKaRa). Thirty micrograms of total protein was separated by 10% SDS-polyacrylamide gel electrophoresis gels, then proteins were transferred to polyvinylidene difluoride membranes. After being blocked with 5% milk for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C, and the appropriate horseradish peroxidase-conjugated secondary antibody was followed. After all, all protein bands were detected by BioRad luminescent imaging system with ECL Chemiluminescent Kit (Thermo Fisher). The primary antibodies used in this study are as follows: anti-Bax (1:1,000; Cat. A0207, ABclonal, Wuhan, China), anti-Bcl-2 (1:1,000; Cat. A0208, ABclonal), anti-Caspase3 (1:1,000; Cat. 9662, Cell Signaling Technology, Boston, MA), anti-CD63 (1:1,000; Cat. A5271, ABclonal), anti-PDCD4 (1:1,000; Cat. AB38428, Absci) and anti-β-actin (1:1,000; Cat. AC026, ABclonal).
10
Penile Tissue Protein Analysis
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The collected penile specimens were lysed in RIPA buffer (Cell Signaling Technology, USA) containing a protease inhibitor cocktail (KeyGEN BioTECH, China). The proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). After blocking, the membrane was incubated with primary antibodies against desmin (Abcam, USA), SM-MHC (Abcam, USA), Collagen1 (Abcam, USA), MMP2 (Abcam, USA), p-SMAD2 (Abcam, USA), SMAD2 (Abcam, USA), and β-actin (Abcam, USA). After washing the membrane three times, appropriate secondary antibodies were applied and incubated with the membrane for 1 h. Protein bands were detected by ECL chemiluminescence reagents (KeyGEN BioTECH, China) and imaged with an imaging system (UVP GDS-8000, USA).
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