After the animals were euthanized, the homogenized hippocampal CA1 (10 mg) was immersed in 300 μL of lysis buffer (Abcam) and 3 μL of butylated hydroxytoluene (Abcam) and centrifuged at 12,000× g at 4°C for 5 min. To determine the MDA level in the hippocampus, LMDA (MDA nmol/mg tissue), 50 μL of the supernatant in a 96-well plate was allowed to react with 150 μL of thiobarbituric acid (Abcam) at 95°C for 1 h and analyzed using the ELISA spectrophotometer at 532 nm at 25°C with calibration of the MDA standard (Abcam). In addition, the homogenized hippocampal CA1 (5 mg) was treated with 100 μL of lysis buffer (Abcam), 100 μL of tissue protein extraction reagent (Thermo Fisher Scientific), and 1 μL of Halt protease inhibitor cocktail (Thermo Fisher Scientific) and centrifuged at 2,000× g at 4°C for 10 min. To determine the AChE activity in the hippocampus, AAChE (mU/mg tissue), 50 μL of the supernatant in a 96-well plate was treated with 50 μL of acetylthiocholine (Abcam) and 50 μL of 0.1% bovine serum albumin (BSA; Abcam) at 25°C for 30 min and analyzed using the ELISA spectrophotometer at 410 nm with calibration of the AChE standard (Abcam) and 0.1% BSA.
Lysis buffer
Manufactured by Abcam
Sourced in United States, United Kingdom
Lysis buffer is a solution used to disrupt cells and release their contents, including proteins, nucleic acids, and other cellular components. It is a fundamental tool in various biological and biochemical applications, such as protein extraction, gene expression analysis, and sample preparation for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Thermo Fisher Scientific (4 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Thiobarbituric acid, by Abcam (2 mentions)
Bis tris polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Lysis buffer, by Thermo Fisher Scientific (2 mentions)
Hif 1α and hydroxy hif 1α, by Abcam (2 mentions)
β actin, by Cytiva (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti il 1β af 401 sp, by R&D Systems (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Anti β actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti irak1 antibody, by Cell Signaling Technology (1 mentions)
Anti rabbit peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Bicinchoninic acid protein concentration kit, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Beyotime (1 mentions)
24 protocols using lysis buffer
1
Hippocampal MDA and AChE Quantification
2
Autophagy Protein Expression Analysis
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HCC827 and NCI-H1650 cells were harvested and lysed in the lysis buffer (Abcam, Cambridge, USA) containing PMSF and protease inhibitor (Roche, USA) for 15 min on ice. Afterward, the cell lysate was centrifuged at 12.000 g at 4°C for 10 min. The protein concentrations were calculated using the BCA Protein Assay kit (Sigma-Aldrich, MO, USA). An equal amount of protein was loaded and electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred onto the polyvinylidene fluoride membrane (Millipore, Billerica, USA). Posttransfer, the membrane was blocked in 5% nonfat milk for one hour at room temperature. Then, the membranes were probed with primary antibodies against DRAM1 (1/2000; ab208160), SQSTM1 (1/10000; ab109012), Beclin-1 (1/2000; ab207612), LC3 (1/2000; ab192890), and GAPDH (1/2500; ab9485) overnight at 4°C, and incubated with the HRP conjugated secondary antibody IgG (1/10000; ab98624) for 1.5 h at room temperature. All antibodies were purchased from Abcam (Cambridge, USA).GAPDH polyclonal antibody was used as an internal control. The protein bands were visualized using an enhanced chemiluminescence visualization system (ECL Plus, Amersham Life Sciences).
3
Western Blot Analysis of Immune Mediators
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Mouse lung samples and BMDMs were homogenized with lysis buffer (Abcam) supplemented with protease and phosphatase inhibitors (78442, ThermoFisher), and the proteins were quantified. Samples were separated by SDS-PAGE and transferred to PVDF membranes using a Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were blocked with skim milk for 2 h, and then probed overnight with anti-GBP5 (gtx31537, Gentex, Zeeland, MI, USA), anti-iNOS (NB300-605, Novus International, St. Louis, MO, USA), anti-IL-1β (AF-401-SP, R&D Systems, Minneapolis, MN, USA), or anti-β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA). After washing with TBST, the blots were incubated with the appropriate anti-mouse, anti-goat, or anti-rabbit secondary antibody and then detected with ECL solution (GenDEPOT, Barker, TX, USA). Semi-quantitative densitometry was performed using ImageJ 2.1.4.6 software (National Institutes of Health, Bethesda, MD, USA).
4
Serum and Pancreatic Insulin Analysis
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Following treatment with SOG for 2 weeks, the mice were fasted for 12 h with free access to water. A total of 50 µl serum was obtained from whole blood by centrifugation for 5 min at 10,000 × g at 4°C and serum fasting insulin of mice was measured using a commercial insulin ELISA kit (Abcam; cat. no. ab100578), according to manufacturer's protocol. In addition, the pancreas were treated with lysis buffer (Abcam; cat. no. ab156035) for 10 min and centrifuged at 4°C for 5 min (10,000 × g) and then the pancreatic levels of insulin and IL-6 in were also detected using insulin and IL-6 ELISA kits (Abcam; cat. no. ab100712), respectively.
5
Western Blot Analysis of IRAK1 Expression
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Pituitaries (n = 6 per group) and inguinal lymph nodes were homogenized in lysis buffer (Abcam, Cambridge, MA, USA) by mortar and pestle in liquid nitrogen. Lysates were centrifuged to remove debris and quantified by a BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Cleared lysates (60 μg/lane) were separated by a reducing SDS–PAGE and transferred to a nitrocellulose membrane. After blocking with 3% bovine serum albumin in PBST (PBS containing 0.05% Tween-20 (VWR-Amresco, Radnor, PA, USA)), the membranes were incubated with an anti-IRAK1 antibody (1:1000, Cell Signaling, Danvers, MA, USA), followed by a peroxidase-conjugated anti-rabbit secondary antibody (1:20,000, Jackson ImmunoResearch, West Grove, PA, USA). The blots were developed by adding a chemiluminescent substrate (Thermo Scientific), and chemiluminescence images were recorded by a CCD imaging system (Hansor, Taichung, Taiwan). The images of IRAK1 and β-actin blots on the same membrane were quantified with the Image J software (version 13.06 for Mac OS X, 64 bit, free software, National Institutes of Health, Bethesda, MD, USA (accessed on 25 August 2021)). Signal intensities of IRAK1 were divided by the intensities of β-actin to obtain normalized IRAK1 expression.
6
Erythrocyte Lysis Protocol
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Red cell lysates were prepared using commercially available lysis buffer (Abcam, Cambridge, UK). According to the manufacturer's instructions, 20 volume units of 1X lysis buffer were mixed with 1 volume unit of whole blood and incubated for 10 minutes at room temperature. The mixtures were then centrifuged at 400x for 5 minutes and the sediments were resuspended in phosphate buffer saline (PBS).
7
Quantification of Protein Expression
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Protein levels were measured as previously described [24 ]. At 48 h post-transfection, 5–8 F and SUNE-1 cells were lysed using the radioimmunoprecipitation assay buffer (lysis buffer; Abcam, China). We quantified the supernatants using a bicinchoninic acid protein concentration kit (Beyotime, China). Subsequently, proteins (20 µg) were separated with 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and were electro-transferred onto a polyvinylidene fluoride membrane. The membrane was incubated with 5% skimmed milk for 1 h at room temperature before being incubated with rat anti-CSE1L (1:1000, Cat. no. ABIN560494, Beijing 4A Biotech Co, Ltd., China), and rat anti-GAPDH (1:1000, Cat. no. AF1186, Beyotime, China) at 4°C for 24 h. The following day, the blot was probed with a mouse anti-rabbit horseradish peroxidase–conjugated secondary antibody (1:1000, Cat. no. 4060–01, Southern Biotech, USA), and protein bands were visualized by chemiluminescence using ECL (Beyotime, China).
8
Oxidative Stress Analysis in HPAEpiCs
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The assays regarding the detection of Malondialdehyde (MDA) and Superoxide Dismutase (SOD) activity were performed to analyze oxidative stress. For lipid peroxidation MDA assay, about 2 × 106 HPAEpiCs were harvested after LPS treatment and/or cell transfection. The cells were washed using PBS and lysed using MDA lysis buffer (Abcam, Cambridge, MA, USA). Cell supernatant was collected by centrifugation, and incubated with thiobarbituric acid (Abcam) at 95 °C for 1 h, followed by analysis using a microplate reader (Thermo Fisher). For SOD activity assay, HPAEpiCs were collected and lysed using lysis buffer (Abcam), followed by centrifugation to harvest cell supernatant. The following assay was performed inferring to the instruction of a SOD activity assay kit (Abcam). The output of samples was determined on a microplate reader (Thermo Fisher).
9
Pharmacological Effects in NMRI Mice
10
Western Blot Protein Analysis Protocol
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Crushed tissues were homogenized in lysis buffer (ThermoFisher) on ice for 1 min (20–30 strokes) using an automatic Dounce homogenizer set to 2000 RPM. Whole cell lysate was prepared in lysis buffer (Abcam) using 3 sets of 3-s sonication (power level 3) intervals on ice. Protein concentration was estimated using Pierce BCA assay kit and samples were prepared and heated for 90C for 5 min. Equal amounts of protein were loaded and separated on 4%–12% Bis-Tris polyacrylamide gel (Thermo Fisher Scientific), transferred to a PVDF membrane, blocked for 1–2 h using 5% fat free skim milk, washed and finally, probed with corresponding antibodies as specified below. Antibodies were from Cell Signaling Technology (HIF-1α and hydroxy HIF-1α dilution 1:3000, MCU dilution 1:5000, MICU1 dilution 1:3000), Abcam (CPT-1a, CPT-2, OXPHOS cocktail dilution 1:3000), ZYMED Laboratories (β-actin; dilution 1:1,000), and Amersham (secondary antibodies conjugated with peroxidase). Development was done using X-ray film using a series of timed exposures and ImageJ was used for densitometric analysis on scanned film images.74 (link)
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