Bovine fibrinogen, thrombin, aminocaproic acid, L-Ascorbic acid and glycerol 2-phosphate were purchased from Sigma (St. Louis, MO). Rat tail type I collagen was purchased from BD Bioscience (San Jose, CA). Fetal bovine serum (FBS), HEPES buffer solution, and penicillin-streptomycin solution were from GibcoBRL (Carlsbad, CA), and ascorbic acid-free α-MEM was from WelGene (Daegu, Korea). The MicroBCA assay kit was from Pierce-Thermo (Rockford, IL). Qunat-iT PicoGreen dsDNA-assay kit was from Invitrogen (Eugene, OR). West-Zol was from Intron Biotechnology (Seoul, Korea). A Dual-GloTM Luciferase Assay System was from Promega (Madison, WI). Protease inhibitor cocktail tablets (Complete) were from Roche (Basel, Switzerland). Anti-Runx2, anti-actin, and HRP-conjugated IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-fibronectin and anti-vitronectin antibodies were from Chemicon (Temecula, CA).
West zol
Manufactured by iNtRON Biotechnology
Sourced in Cameroon
West-Zol is a reagent used in the process of Western Blotting, a widely used analytical technique in molecular biology and biochemistry. It is designed to extract and solubilize proteins from various biological samples, such as cell lysates or tissue homogenates, for subsequent electrophoretic separation and detection.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Aminocaproic acid, by Merck Group (1 mentions)
Bovine fibrinogen, by Merck Group (1 mentions)
Anti fibronectin, by Merck Group (1 mentions)
Anti runx2, by Santa Cruz Biotechnology (1 mentions)
Dual glotm luciferase assay system, by Promega (1 mentions)
Protease inhibitor cocktail tablets complete, by Roche (1 mentions)
Hepes buffer solution, by Thermo Fisher Scientific (1 mentions)
Thrombin, by Merck Group (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Glycerol 2 phosphate, by Merck Group (1 mentions)
Rat tail collagen type 1, by BD (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Anti phospho p44 42 mapk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Lipofectamine plus, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Anti actin sc 1616, by Santa Cruz Biotechnology (1 mentions)
8 protocols using west zol
1
Osteogenic Differentiation of Mesenchymal Cells
2
Western Blot for BRAF, KSR1, and Notch1
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BCPAP and 8505C cells were cultured in RPMI media (Sigma-Aldrich) and HeLa cells were grown in DMEM (Sigma-Aldrich). The cells were transiently transfected with pEFm-BRAFV600E or SiKSR1 (sc-35762, Santa Cruz Biotechnology) using Lipofectamine PLUS (Invitrogen). The cells were lysed in lysis buffer, and the lysates were separated by SDS-PAGE. After proteins were transferred to a nitrocellulose membrane (Amersham Biosciences), the membranes were blocked with 5% skimmed milk and incubated with the indicated primary antibodies overnight at 4 8C; after washing, the membranes were incubated with secondary antibodies for 1 h at room temperature. The immunoreactive bands were developed using peroxidase-conjugated secondary antibodies (WEST-ZOL, iNtRON Biotechnology, Kyungki-Do, South Korea). The primary antibodies used in this study were anti-BRAF (F-7) (sc-5284, Santa Cruz Biotechnology), anti-KSR1 (sc-25416, Santa Cruz Biotechnology), anti-Notch1 (#4380, Cell Signaling, Danvers, MA, USA), anti-phospho-p44/42 MAPK (Thr202/Tyr204) (#4376, Cell Signaling), and anti-actin (sc-1616, Santa Cruz Biotechnology).
3
SHE and STAT5 Inhibition in Murine Splenocytes
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Murine splenocytes were incubated with SHE (0, 62.5 and 125 µg/mL in PBS) in the presence or absence of OVA (100 µg/mL in PBS) for 24, 48, and 72 h. For inhibitor experiments, the STAT5 inhibitor (CAS 285986-31-4) suspended in PBS was added 2 h prior to the OVA and SHE treatments with a 24 h incubation period. Cytoplasmic and nucleic proteins were extracted from the cells by use of the NE-PER Nuclear and Cytoplasmic Extraction Regents kit (Thermo Fisher Scientific, Walthman, MA). After measuring the protein concentration using a Bio-Rad Assay (Bio-Rad, Hercules, CA), the proteins (40 µg/mL) were separated using 7.5% SDS-PAGE and immunoblotted on nitrocellulose membranes (Bio-Rad, Hercules, CA).
The membranes were incubated with 2% skim milk followed by another round of incubation with the primary antibodies: STAT5, p-STAT5, NLRP3 (Cell Signalling Technology, Inc.), and β-actin (Sigma, St. Louis, MO, USA) overnight. After washing, the membranes went through a final round of incubation with an appropriate horseradish peroxidase (HRP)-conjugated anti-mouse Immunoglobulin G (IgG) (Sigma, St. Louis, MO) or anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) for 45 min. The blots were developed with WestZol (iNtRON Biotechnology, Sungnam, Korea), and the densitometric analysis was performed with ImageJ software (version 1.47).
The membranes were incubated with 2% skim milk followed by another round of incubation with the primary antibodies: STAT5, p-STAT5, NLRP3 (Cell Signalling Technology, Inc.), and β-actin (Sigma, St. Louis, MO, USA) overnight. After washing, the membranes went through a final round of incubation with an appropriate horseradish peroxidase (HRP)-conjugated anti-mouse Immunoglobulin G (IgG) (Sigma, St. Louis, MO) or anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) for 45 min. The blots were developed with WestZol (iNtRON Biotechnology, Sungnam, Korea), and the densitometric analysis was performed with ImageJ software (version 1.47).
4
Cytokine Signaling Pathway Assay
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LDH Cytotoxicity Detection Kit was purchased from Takara Bio Inc., (Kusatsu, Japan). HEPES buffer, hydrocortisone, β-estradiol, chloroform, and β-actin were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Insulin-Transferrin-Selenium, l -glutamine, fetal bovine serum, Dulbecco’s modified eagle’s medium/nutrient mixture F-12 (DMEM/F12), and Trizol were purchased from Gibco Life Technologies (Grand Island, NY, USA). 3H-thymidine was purchased from Amersham Life Science (Arlington Heights, IL, USA). Primary antibodies for p38, phospho p38, ERK1/2, pERK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA), and those for JNK, pJNK were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Westzol was purchased from iNtRON Biotechnology (Sungnam, Korea). cDNA synthesis kit was purchased from Promega (St Louis, MO, USA). Power SYBER Green PCR Master Mix was purchased from Applied Biosystems (Foster City, CA, USA). Mouse IL-1β, IL-6 ELISA kits were purchased from BioLegend (San Diego, CA, USA). Mouse IL-33 ELISA kit was purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). Ethanol was purchased from OCI Chemicals (Seoul, Korea).
5
Heparin Binding Affinity Assay
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The binding affinity of heparin to the peptides was measured via a slot-blot assay. Various concentrations of HBP in phosphate-buffered saline (PBS) were immobilized onto a nitrocellulose membrane in the slot-blot wells, and the wells were blocked with Tris-buffered saline (TBS) with 0.5% skim milk for 1 hour. Then, heparin (20 mg/mL) was incubated in the slot-blot wells for 90 minutes. The wells were then incubated overnight at 4°C with a mouse antiheparin antibody, followed by a reaction with horseradish peroxidase (HRP)-conjugated HBP (Lifespan Technologies, Salt Lake City, UT, USA) in PBS for 1 hour at room temperature. The bound antibodies were detected using chemiluminescence reagents (West-Zol; Intron Biotechnology, Seongnam, South Korea).
6
Identification of Endogenous Chaperones in E. coli
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Endogenous chaperones in the E. coli lysates were prepared and identified by western blot. After electrophoresis, the gel was transferred to PVDF membranes (Thermo Fisher Scientific) by using iBlot2 Transfer Stacks and iBlot2 Dry Blotting System. The membranes were blocked with 5% skim milk in TBST (20 mM Tris, 137 mM NaCl, 2.7 mM KCl, and 0.1% Tween-20) for 1 hour and then washed 3 times with TBST buffer. The blocked membranes were incubated with primary antibodies α-TF (M201, Takara), α-DnaK (ab69617, Abcam), α-DnaJ (ADI-SPA-410, Enzo), α-GroEL (ab82592, Abcam), and α-GroES (ab69823, Abcam) diluted in TBST overnight at 4°C. The membranes were washed 3 times in TBST, then incubated with a secondary antibody—α-mouse or α-rabbit IgG Ab conjugated with horseradish peroxidase (Sigma), depending on the origin of the first antibody – diluted 1:20,000 in TBST, for 40 minutes, and washed 3 times in TBST. The membranes were reacted with an ECL mixture using WEST-ZOL (Intron Biotechnology) and exposed to an X-ray film in a dark room.
7
Western Blot Analysis of Melan-a Cells
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Melan-a cells were treated with different concentrations of GD for the indicated times. The cells were lysed with extraction buffer, and the protein concentrations were determined using the BCA method. The proteins from the cell lysates were loaded onto 10% SDS-polyacrylamide gels and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). Nonspecific binding was blocked with 5% BSA in TBS containing 0.01% Tween-20 for 1 h at room temperature before incubation with diluted primary antibodies (1:1000 or 1:500) overnight at 4 °C. Then, the membranes were incubated with specific secondary antibodies (1:1000) for 1 h and washed three times with TBS-Tween 20. The expression of β-actin was used as an internal standard. Membranes were subjected to WestZol (iNtRON Biotechnology, Gyeonggi-do, Korea) and were visualized using the LAS-4000 imaging system (Fuji film Corp., Tokyo, Japan).
8
Nuclear and Cytoplasmic Protein Extraction
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The H9-treated cells were collected and fractionated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific Inc, IL, USA), according to the manufacturer's protocol. After fractionation, we calculated each extract concentration, performed a 15% Tris-glycine gel electrophoresis, and transferred them to a PVDF membrane. The membrane was blocked with 5% TBST skim milk and incubated with primary antibodies overnight. The membrane was washed with TBST and incubated with secondary antibodies for 1 h. After washing with TBST, we added a chemiluminescent substrate (Westzol; iNtRon Biotechnology).
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