Mice were perfused with PBS containing heparin (5 U/mL; Sigma; H-3125) solution. Spinal cords were dissected and flash frozen. Frozen spinal cords were homogenized in 1 mL of PBS with protease inhibitor (MedChem Express; HY-K0010) using a Dounce homogenizer. Triton X-100 was added to final concentration of 0.2% and samples were vortexed. Samples were centrifuged at 10,000 g for 5 min at 4°C and 200 μL was acquired from the top layer to be flash frozen for later analysis. Mouse TH17 Luminex was run per manufacturer’s instructions on a MagPix by the University of Virginia Flow Cytometry Core, RRID: SCR_017829.
Hy k0010
Manufactured by MedChemExpress
Sourced in United States, China
HY-K0010 is a lab equipment product manufactured by MedChemExpress. It is designed for the purpose of performing laboratory tasks. The core function of this product is to assist researchers and scientists in their work within the laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Hy k0022, by MedChemExpress (9 mentions)
A600669, by Sangon (5 mentions)
Ipvh00010, by Merck Group (4 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Hy k0021, by MedChemExpress (2 mentions)
Microplate reader, by Molecular Devices (2 mentions)
Hy k0023, by MedChemExpress (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Luminata crescendo western hrp substrate, by Merck Group (2 mentions)
Bca protein assay kit, by Dingguo (2 mentions)
Luminata crescendo immunoblotting hrp substrate, by Merck Group (2 mentions)
C3117, by Merck Group (2 mentions)
Ai600 imaging system, by GE Healthcare (2 mentions)
P0013, by Beyotime (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by MedChemExpress (2 mentions)
H 3125, by Merck Group (1 mentions)
Ab180948, by Abcam (1 mentions)
Ab283264, by Abcam (1 mentions)
T55561, by Abmart (1 mentions)
25 protocols using hy k0010
1
Analyzing Mouse TH17 Cells via Luminex
2
Signaling Pathway Analysis of hESCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from human endometrial stromal cells (hESC) and rat uterine tissues using ice-cold RIPA lysis buffer (WB3100, NCM Biotech) supplemented with a mix of protease and phosphatase inhibitors (HY-K0010, HY-K0021, HY-K0022, MedChem Express). Protein concentrations were quantified with a BCA Protein Assay Kit (WB6501, NCM Biotech). Equal amounts of the protein lysates were resolved on 10% SDS-PAGE gels (P2012, NCM Biotech) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010, Millipore). The membranes were blocked with 5% nonfat milk for one hour at room temperature, then incubated with primary antibodies targeting IGFBP1 (ab180948, Abcam), AKT (T55561, Abmart), phospho-AKT (Ser473) (Cell Signaling Technology Cat# 4060), NR4A1 (ab283264, Abcam; T56890, Abmart), and β-ACTIN (ACTB) (GB15003, Servicebio) overnight at 4 °C. This was followed by a one-hour room temperature incubation with the appropriate secondary antibodies. Band detection was performed using an ECL detection system (P10100, NCM Biotech), and band intensities were analyzed and normalized to ACTB using ImageJ software. The phosphorylation status of AKT was determined by calculating the ratio of phosphorylated AKT (Ser473) to total AKT.
3
Western Blot Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease and phosphatase inhibitor cocktail (HY-K0010 and HY-K0022; MedChemExpress) on ice for 15 min. Protein samples were separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). After being blocked in 5% skim milk (A600669; Sangon Biotech) at room temperature for 2 h, the membrane was incubated with the primary antibody overnight at 4°C and then with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. The target protein bands were detected with the ECL enhanced chemiluminescence detection system (Tanon 6200 chemiluminescence imaging workstation; Tanon Science & Technology Co., Ltd., Shanghai, China). The bands were then quantified by densitometry using ImageJ software.
4
Liver Tissue Cytokine Extraction and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The extraction method of liver tissues was described by Chen et al. [58 (link)]: 0.5 g of liver tissues was homogenized in 1.5 mL of ice-cold buffer containing 50 mM Tris (pH 7.2), 150 mM NaCl, 1% Triton X-100, and 0.1% protease inhibitor (PI) (HYK0010, MedChemExpress, Monmouth Junction, NJ, USA). The homogenized solution was then centrifuged at 3000 rpm for 15 min at 4 °C, and the supernatant was collected. Concentrations of hepatic TNF-α, IL-1β, IL-6, and IL-10 were determined by corresponding enzyme-linked immunosorbent assay (ELISA) kits, including rat TNF-α (BioLegend Systems, San Diego, CA, USA), IL-1β (Rat IL-1, R&D Systems, Minneapolis, MN, USA), IL-6 (Rat IL-6, R&D Systems) and IL-10 (Rat IL-10, R&D Systems, Minneapolis, MN, USA). The OD was read at 450 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA) for all cytokines.
5
Comprehensive Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sample tissues and cultured cells were lysed with RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor cocktails (HY-K0010, MedChemExpress, Shanghai, China) and phosphatase inhibitor cocktails (HY-K0023, MedChemExpress, Shanghai, China), then centrifugated at 15,000 g for 15 min at 4 °C. The protein concentration was measured using the BCA protein assay kit (Thermo Fisher, MA, USA). The proteins were denatured at 100 °C for 5 min in SDS-PADE protein buffer (Beyotime, Shanghai, China), and separated on 7.5–15% SDS–polyacrylamide gels. After the cells were incubated in serum-free medium for 24 h at 37 °C, the conditioned medium (CM) was collected by centrifugation at 2,000 g for 10 min at 4 °C. TCA (T0699, Sigma-Aldrich, USA) precipitation was performed to collect the secreted proteins in the CM, as previously described [24 (link)]. The antibodies used in WB are listed in Supplementary Table 4 .
6
Western Blot and Immunoprecipitation Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed three times with cold PBS and lysed with NP‐40 buffer supplemented with a cocktail protease inhibitor (HY‐K0010, MedChemExpress) for 30 min at 4 °C. The protein concentrations were measured using a bicinchoninic acid assay kit (#23 225, Thermo Fisher Scientific). The proteins were then electrophoresed in SDS‐PAGE gels and transferred onto polyvinylidene difluoride membranes. After blocking with 5% non‐fat milk, the membranes were incubated with primary antibodies overnight at 4 °C. After incubation with 1:5000 horseradish peroxidase‐linked secondary antibodies at 25 °C for 2 h, the membranes were visualized using an efficient chemiluminescence kit (#34 096, Thermo Fisher Scientific).
For IP, proteins were extracted as described for the Western blot assay. After centrifugation, the supernatant was incubated with magnetic beads (HY‐K0205; MCE) overnight at 4 °C. The proteins were eluted from the magnetic beads and collected for subsequent experiments.
For IP, proteins were extracted as described for the Western blot assay. After centrifugation, the supernatant was incubated with magnetic beads (HY‐K0205; MCE) overnight at 4 °C. The proteins were eluted from the magnetic beads and collected for subsequent experiments.
7
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were lysed in RIPA buffer (P0013B, Beyotime Biotechnology) supplemented with a protease and phosphatase inhibitor cocktail (HY-K0010 and HY-K0022, MedChemExpress). The protein concentration was determined using a BCA Protein Assay Kit (BCA01, DingGuo). The protein samples were separated by SDS‒PAGE and transferred to a membrane. The membrane was incubated with 5% skim milk (A600669, Sangon) for 1 h at room temperature. The membrane was then incubated with the primary antibody overnight at 4 ℃, followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Luminata Crescendo Western HRP substrate (WBLUR0500, Millipore) was used to visualize the immunoblotting results via a GE AI600 imaging system.
8
Western Blot Analysis of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
WB was performed to confirm the expression of recombinant proteins. Cells (∼106 cells per well) were lysed with 60 μl of radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride [HY-B0496; MedChemExpress], and protease inhibitor cocktail [HY-K0010; MedChemExpress]) on ice for 30 min. After incubation, the sample was centrifuged at 21,600 × g for 10 min at 4°C to remove the insoluble fraction. The supernatant was mixed with SDS-PAGE loading buffer and boiled at 95°C for 5 min or kept at 4°C overnight (for multitransmembrane proteins). The proteins were separated by SDS-PAGE and transferred onto poly vinylidene di-fluoride membranes, and the membranes were blocked at RT in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and 5% nonfat milk for 1 h, followed by incubation with primary antibodies at RT for 1 h. After washing three times with TBS-T buffer, the membranes were then incubated with HRP-labeled secondary antibodies for 1 h at RT. After washing with TBS-T, the membrane was visualized with ECL Prime WB Detection Reagent (GE Health care).
9
Gastrocnemius Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Right gastrocnemius muscle tissues were cut into 0.1 g pieces and homogenized in 0.4 mL RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, and 1% NP-40 at pH 7.5) containing 1% of a PI (HYK0010, MedChemExpress, Monmouth Junction, NJ, USA) and phosphatase inhibitor (PPI) (HYK0022, MedChemExpress, Monmouth Junction, NJ, USA). After an ice bath for 30 min, the homogenate was centrifuged at 10,000× g for 10 min at 4 °C. The target proteins were analyzed by western blotting described as the previous studies [53 (link),54 (link)]. The antibodies were listed in Table 8 .
10
Measuring Tight Junction Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Right gastrocnemius muscle tissue (0.1 g) was homogenized in 0.4 mL RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, and 1% NP-40 at pH 7.5) containing 1% of a PI (HYK0010, MedChemExpress, Monmouth Junction, NJ, USA) and phosphatase inhibitor (PPI) (HYK0022, MedChemExpress, Monmouth Junction, NJ, USA). After an ice bath for 30 min, the homogenate was centrifuged at 10,000× g for 10 min at 4 °C. The target proteins were analyzed by western blotting, described as the tight junction protein level measurement. The antibodies are listed in Supplementary Table S5 .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!