The spinal cord tissues of the rats or the cell samples were then lysed with lysis reagent (Beyotime, Shanghai, China). A BAC protein assay kit (Thermo Scientific, Rockford, IL, USA) was used to quantify the proteins. Western blot was then routinely performed by 10% sodium dodecyl sulfate (SDS) gels and polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Bedford, MA, USA). An Odyssey Infrared Imaging system (Biosciences, Lincoln, NE, USA) was used to detect the blotted membrane. Antibodies against HSPB8 (R&D Systems, Minneapolis, MN, USA), LC3B (Cell Signaling Technology, Beverly, MA, USA), BAG3, Beclin-1, p62, and GADPH (ProteinTech Group, Chicago, IL, USA) were adopted. Secondary antibodies were obtained from LI-COR Biosciences (Lincoln, NE, USA).
Bac protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BAC protein assay kit is a laboratory tool used to quantify the total protein content in a sample. It provides a simple and reliable method for determining the protein concentration in biological samples. The kit utilizes a bicinchoninic acid (BCA) reagent to produce a colorimetric reaction, allowing for the measurement of protein levels using a spectrophotometer.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Lysis reagent, by Beyotime (1 mentions)
Gadph, by Proteintech (1 mentions)
Beclin 1, by Proteintech (1 mentions)
Modulyod, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence substrate, by Boster Bio (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
Chemidoc xrs system with image lab software, by Bio-Rad (1 mentions)
Ab185627, by Abcam (1 mentions)
Ab124762, by Abcam (1 mentions)
Transilluminator, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Ab65081, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Roche (1 mentions)
Pyruvate kinase activity assay kit, by Merck Group (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Qproteome nuclear protein kit, by Qiagen (1 mentions)
7 protocols using bac protein assay kit
1
Quantifying Protein Expressions in Rat Spinal Cord and Cells
2
Serum Protein Depletion and Quantification
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PierceTM Top 12 abundant protein depletion Spin column (Thermo scientific, UK) was used to deplete OA patient serum sample as per manufacturer’s recommendations. Depleted serum was then concentrated using a freeze-dryer (MODULYOD, Thermo electron corporation). Samples were re-suspended in 50μl of deionised water and quantified using a bicinchoninic acid (BAC) protein assay kit (Fischer Scientific, UK) according to the manufacturer’s guidelines. Equal volumes (15μl) of protein sample were mixed with sample buffer (1M Tris-HCl, 10% sodium dodecyl sulfate (SDS), 40% glycerol, 0.5% Coomassie blue, and 2% 2-mercaptoethanol (Sigma, UK)). Mixed samples were boiled at 95°C for 5min and vortexed. Samples were then loaded into wells of SDS-PAGE for western-blotting.
3
Quantitative Immunoblotting for Protein Analysis
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Total protein was extracted from cells and quantified using the BAC Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Proteins (20 μg per well) were resolved on 12% SDS-polyacrylamide gels and transferred onto PVDF membranes (Millipore, Boston, MA, USA). Transferred membranes were blocked in 5% fat free milk at room temperature for 1 h, and then incubated with primary antibodies, according to the manufacturer’s instructions. Then the membranes were washed three times in TBST, and incubated with correspondent secondary antibodies at room temperature for 1 h. Immunoreactive proteins were visualized using enhanced chemiluminescence substrate (BOSTER, Wuhan, China) and ChemiDoc™ XRS+ System with Image Lab™ Software (BIO-RAD, Hercules, CA, USA). β-Actin antibodies (Cell Signaling Technology, Boston, MA, USA) were used as a control.
4
Western Blot Analysis of Protein Expression
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Proteins from LSCs were extracted with a cold lysis buffer composed of protease and phosphatase inhibitors (ThermoFisher Scientific, Waltham, MA, USA). Protein concentration was measured by a BAC protein assay kit (ThermoFisher Scientific, Waltham, MA, USA). Equal amounts of protein extracts (15 μg) were subjected to electrophoresis on 10% acrylamide gels and then transferred to polyvinylidene difluoride membranes (Roche, Basel, Switzerland). After being blocked in 5% BSA for 1 h at 25 °C, the membranes were incubated overnight at 4 °C with primary antibodies for anti-p63 (1:1000 dilution, ab124762, Abcam, Cambridge, UK), C/EBPδ (1:1000 dilution, ab65081, Abcam, Cambridge, UK), and K12 (ab185627, Abcam, Cambridge, UK). After 3 washes with Tris-buffered saline containing 0.05% Tween-20 for 10 min each, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (1: 5000 dilution, 31460, ThermoFisher Scientific, Waltham, MA, USA) for 1 h at room temperature. An HRP-conjugated mouse anti-β-actin antibody (1:1000 dilution, A3854, Sigma-Aldrich, Saint Louis, MO, USA) was used for protein quantification. The results were detected by an enhanced chemiluminescence reagent kit (NCM Biotech, Suzhou, China) and recorded by the transilluminator (Bio-Rad, Hercules, CA, USA).
5
Measuring Pyruvate Kinase Activity
6
Western Blot Analysis of Oxidative Stress Markers
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Isolated MGs were extracted in a cold lysis buffer composed of protease and phosphatase inhibitors. Protein concentration was measured by BAC protein assay kit (Cat #23225; ThermoFisher Scientific). Three samples were used in each group. Equal amounts of protein extracts (20 µg) were subjected to electrophoresis on 10% acrylamide gels and then electronically transferred to polyvinylidene difluoride (PVDF) membranes. After blocking in 5% BSA for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies for anti-PPARγ (1:500, 2 µg/mL), p-ERK1/2 (1:1000, 0.084 µg/mL), ERK1/2 (1:1000, 0.191 µg/mL), NF-κB p65 (1:1000, 0.208 µg/mL), phospho-NF-κB p65 (1:1000, 0.057 µg/mL), Nrf2 (1:1000, 0.7 µg/mL), HO-1 (1:1000, 1 µg/mL), SOD2 (1:1000, 1 µg/mL), Tom20 (1:1000, 0.4 µg/mL), Tim23 (1:1000, 0.4 µg/mL), AMPKα (1:1000, 0.2 µg/mL), and phospho-AMPKα (1:1000, 0.027 µg/mL). After 3 washes with Tris buffered saline containing 0.05% Tween-20 for 10 minutes each, the membranes were incubated with HRP-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies. HRP-conjugated mouse anti–β-actin was used for protein quantification. The results were detected by enhanced chemiluminescence reagent (ECL-500; ECL, Lulong Inc., Xiamen, China) and recorded by the transilluminator (ChemiDoc XRS System; Bio-Rad, Philadelphia, PA, USA).
7
Protein Extraction and Western Blotting
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Cells were collected and homogenized with RIPA buffer (Thermo Scientific) containing complete protease inhibitors for protein extraction. For nuclear and cytoplasmic fractionation, the Qproteome Nuclear Protein Kit (QIAGEN) was used. The protein concentration was quantified by a BAC protein assay kit (Thermo Scientific). Cell Lysate was denatured by adding 6 3 sample buffer containing 330 mM Tris-Cl pH 6.8, 30% glycerol,1 mM EDTA, 9% sodium dodecyl sulfate (SDS), 550 mM DTT, 0.3% bromophenol blue and heated for 10 min at 90 C. Proteins were separated on SDS-polyacrylamide gels and processed for conventional western blotting. The primary and secondary antibodies were diluted in TBST (Tris: 20 mM, NaCl: 150 mM, Tween 20 detergent: 0.1% w/v) containing 5% (w/v) skimmed milk. Peroxidase activity was detected by chemiluminescence (Tanon, High-sig ECL Western Blotting Substrate, Cat#180-501) and captured with Amersham Imager 600 (General Electric). The antibodies used are listed in key resources table.
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