Total proteins were obtained from the treated BEND cells with a protein extraction kit (Solarbio, Beijing, China). The protein concentration was detected by the BCA method (Solarbio, Beijing, China). The proteins (50 μg) were heated in loading buffer at 95 °C for 5 min. Each protein sample was subjected to SDS-PAGE using a gel preparation kit (Solarbio, Beijing, China) to separate proteins, then proteins were transferred to the PAGE membrane and blocked with 5% skim milk at 4 °C overnight. After rinsing with TBST on a shaker, membranes were hybridized for 90 min at room temperature with anti-Nrf2 (1:2000, Abcam, Cambridge, UK), anti-GCLC (3:1000, Abcam), anti-HO-1 (3:1000, Abcam), anti-NQO-1 (1:2000, Novus, Centennial, CO, USA), anti-Mn-SOD (1:5000, Abcam), and anti-β-actin (1:1000, Cell Signaling) antibodies, respectively. After rinsing for 1 h, membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies for 90 min at room temperature. After rinsing again for 1 h, proteins were visualized using Enhanced Chemiluminescence (ECL) system. Finally, an image-analysis system was used to analyze the density of these target proteins.
Protein extraction kit
Manufactured by Solarbio
Sourced in China
The Protein Extraction Kit is a laboratory tool designed to efficiently extract proteins from various biological samples. It provides a standardized and reliable method for isolating proteins, a crucial step in many biochemical and analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (10 mentions)
Bca kit, by Solarbio (4 mentions)
Bca protein assay kit, by Solarbio (4 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Trans blot machine, by Bio-Rad (3 mentions)
Anti nrf2, by Abcam (2 mentions)
Anti ho 1, by Abcam (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Gel doc xr gel documentation system, by Bio-Rad (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Anti bax, by Abcam (2 mentions)
Anti bcl 2, by Abcam (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Chemiluminescence imaging system, by Bio-Rad (2 mentions)
Bicinchoninic acid protein assay kit, by Solarbio (2 mentions)
Sds page, by Keygen Biotech (2 mentions)
Goat anti rabbit igg, by Proteintech (2 mentions)
Enhanced chemiluminescence system, by Merck Group (2 mentions)
Goat anti mouse igg, by Proteintech (2 mentions)
57 protocols using protein extraction kit
1
Nrf2 Signaling Pathway Protein Analysis
2
Protein Extraction and Western Blot Analysis
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Cell proteins were extracted with a protein extraction Kit (Solarbio Company, Beijing, China), then mixed with protein loading buffer and denatured at 100 °C for 10 min with 1 mM phenylmethylsulfonyl fluoride (Millipore, Billerica, MA, USA), and 1 mM ethylenediaminetetraacetic acid (EDTA). The membranes were then incubated sequentially with antibodies against GAPDH (rabbit anti-GAPDH, 1:10,000 Abcam, Cambridge, UK) and PLIN2 (rabbit anti-PLIN2, 1:800 Abcam NT, HK) for 12 h at 4 °C. After washing three times (10 min each) with PBS-Tween 20, the membranes were incubated with secondary IgG-Goat anti-Rabbit HRP antibodies (1:2000 NOVUS NT, HK) diluted in PBS-Tween 20 (0.08 μg/mL) for 2 h, and then washed in PBS-Tween 20. Chemiluminescent detection (Millipore) was performed by mixing equal volumes of Luminol Reagent and Peroxide solution in a clean container or test tube and then applying the mixture to PVDF membranes. Immunoreactivity was detected using a Gel Doc™ XR+ Gel Documentation System (Bio-Rad, Hercules, CA, USA).
3
Protein Extraction and Western Blot Analysis
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Proteins were extracted from cells using a protein extraction kit (Solarbio, Beijing, China). Protein concentration was quantified via a BCA assay kit (Thermo Scientific, Waltham, MA, USA). 20 μg of protein samples underwent separation by SDS-PAGE, transferring to PVDF membranes (ISEQ00010, Millipore), and then blocking with 5% bovine serum albumin (BSA, Solarbio). Following this, the membranes were incubated with primary antibodies overnight at 4 °C. Thereafter, HRP-conjugated secondary antibodies were introduced at room temperature for 2 h. Reacting bands were visualized using ECL reagent (170-5061, Bio-Rad, Hercules, CA, USA), with protein band densities semi-quantified via Image J (National Institutes of Health, Bethesda, MD, USA).
4
Western Blot Analysis of Inflammatory Markers
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Total cellular proteins were extracted from cultured cells using the protein extraction kit (Solarbio, Beijing, China). An equal amount of protein (40 μg) from each sample was subjected to SDS-PAGE and then transferred onto polyvinylidenedifluoride (PVDF) membranes (Thermo Fisher). After blocking with 5% skimmed milk at room temperature for 1 h, the membranes were incubated with primary antibodies at 4°C overnight followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies (1:10000, ZSGB-Bio, Beijing, China) for 1 h at room temperature. Finally, the protein bands were imaged with Chemidox XRS (Bio-Rad, Hercules, United States) and quantified with Quantity One software (Bio-Rad). The following primary antibodies were used: LOX-1, ICAM-1, E-selectin, Integrin αM, Integrin β2 (1:1000, Bioss, Beijing, China), CCR2 (1:200, Santa Cruze, Delaware Ave, United States), MCP-1, p65, p–p65 (1:1000, Abcam, Cambridge, United Kingdom) and GADPH (1:1000, Protein, Wuhan, China).
5
Protein Isolation and Western Blot Analysis in Adipocytes
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Total cellular protein was isolated from adipocytes with a protein extraction kit (Solarbio Company, Beijing, China) after mixing with protein loading buffer and denaturation for 10 min in a 100 °C metal bath. A 20-µg protein sample was electrophoresed on a 12% gel, and the protein was transferred to a PVDF membrane after gel electrophoresis. Next, the membrane was incubated with antibodies against β-ACTIN (1:5000, NOVUS, HK, NP_776404.2), TP53INP2 (1:2000, AVIVA Systems Biology, San Diego, CA, USA, XP_003586891.1), PPARγ (1:1000, Boster, Wuhan, China, NP_851367.1), PLIN2 (1:2000, Abcam, Cambridge, UK, NP_776405.1), FASN (1:2000, Abcam, NP_777087.1), p62 (1:2000, Abcam, NP_788814.1), or LC3 (1:2000, Abcam, NP_001001169.1) for 12 h at 4 °C. After washing the membrane three times (10 min each) with PBS-Tween 20, the membrane was incubated with the secondary antibody for 1 h and then washed three times with PBS-Tween 20 (10 min each). Finally, equal amounts of luminol reagent and peroxide solution were mixed in an EP tube and added dropwise to the PVDF membrane. The Gel Doc™ XR+ Gel Documentation System (Bio-Rad, Hercules, CA, USA) was used to detect the immunoreactivity.
6
Western Blot Analysis of Protein Expression
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Protein extraction kit (Solarbio, China) was used to isolate proteins, and then the concentration of the proteins was detected by BCA Protein Assay kit (Cwbio, China). The cell lysate was loaded on 10% SDS-PAGE after being boiled for 5 min to denaturation and then transferred onto a PVDF membrane (Millipore, USA). The non-specific antigens were blocked using 5% skimmed milk. Primary antibodies were incubated at 4 °C overnight. After removing the appropriate horseradish peroxidase-conjugated secondary antibody (ab97051, 1:3000, Abcam, USA), the blots were detected using Enhanced Chemiluminescence Detection kit (GE Healthcare Biosciences). The primary antibodies used in the experiment were as follows: N-cadherin (ab18203, 1:1000), Vimentin (ab137321, 1:1500), anti-PAX8 (ab53940,1:200), anti-p-Erk1/2 (ab201015, 1:1000), anti-Bcl-2 (ab593480, 1:700), anti-Bax (ab53154, 1:1000), anti-Bak (ab32371, 1:10,000) purchased from Abcam. E-cadherin (3195, 1:1000), anti-p-Akt (Thr308) (13,038, 1:1000), p-JNK1/2(4668, 1:2000), anti-GAPDH (5174, 1:1000), anti-β-actin (4970, 1:1000) purchased from Cell Signaling Technology.
7
Protein Expression Analysis by Western Blot
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Proteins were extracted with Protein Extraction Kit (Solarbio, China) and their concentrations were checked using BCA Kit (Solarbio, China). After being separated using SDS-PAGE (Bio-rad, USA), the proteins were transferred to the PVDF membrane (Merck, USA). After blocking, the samples were treated with primary antibodies against LOX-1 (1:1000, ab60178), p-p38MAPK (1:1000, ab4822), p38MAPK (1:1000, ab170099) and β-actin (1:500, ab6276) (Abcam, USA) at 4°C for 12 h. After being rinsed with TBST (Solarbio, China), the secondary HRP-conjugated antibody was added and incubated for 1 h. Next, after TBST washing, the bands were visualized using the DAB kit (Solarbio, China). The relative expression levels of proteins were obtained via Image J 1.53 f (NIH, USA).
8
Quantitative Protein Analysis in Spinal Cord
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The total protein from spinal cord tissues and cell samples was extracted using a protein extraction kit (Solarbio, Beijing, China), following the provided instructions, and the protein concentration was quantified using the BCA method. After gel preparation, the samples were loaded in equal amounts of total protein. The proteins was separated by 10% SDS-PAGE (80 V, 2 h, then 130 V, 1 h) and transferred to a PVDF membrane. The membrane was blocked with 5% milk for 1 h, followed by overnight incubation at 4°C with primary antibodies against NF200 (1:1000, 2,836, Cell Signalling Technology, Beverly, MA, United States), GAP43 (1:1000, 8,945, Cell Signalling Technology, Beverly, MA, United States), PI3K (1:1000, 4,292, Cell Signalling Technology, Beverly, MA, United States), p-PI3K (1:500, AF3242, Affinity, China), Akt (1:1000,bs-6951R, Bioss, Beijing, China), p-Akt (1:1000,bsm-60645R, Bioss, Beijing, China), and GAPDH (1:8000, 2,118, Cell Signalling Technology, Beverly, MA, United States). This was succeeded by incubation with HRP-conjugated secondary antibodies (1:10000, 7,074, 7,076, Cell Signalling Technology, Beverly, MA, United States) for 1 h at room temperature. Lastly, the membranes were visualized using ECL reagents and the Azure Biosystems NIR Fluorescence Imaging system, followed by quantitative grayscale analysis using Image J software.
9
Protein Extraction from Tissue Samples
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Nuclear and cytoplasmic protein extraction was carried out after homogenizing the tissues with a tissue homogenizer. Protein extraction was performed as per the protein extraction kit (Solarbio, China) instructions.
10
Chloroquine Modulates Autophagy in INS-1 Cells
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INS-1 cells (1 × 10 5 /mL) were pretreated with 0 and10 μM chloroquine (CQ, Sigma) 13 (link) ; then treated with 50, 100, and 200 μM α-LA and 0.1 μM rapamycin for 4 h 14 ; and incubated with 30 μM NaAsO 2 for 24 h. At the time of the designated treatments, the cells were washed twice with ice-cold PBS and completely lysed in lysis buffer according to the instruction of the protein extraction kit (Solarbio). The cell lysate was centrifuged at 12,000 × g at 4°C for 10 min, and the supernatants containing total protein were isolated. Total protein concentration was detected by the BCA method. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis was performed and the proteins were transferred to a polyvinylidene membrane. The blots were blocked with 10% non-fat milk, incubated with primary antibodies against LC3B (CST), P62 (CST), phosphatidylinositol 3-kinase (PI3K, CST), and mammalian target of rapamycin (mTOR, CST), and β-actin (Santa Cruz Biotechnology); and then incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature. The color was displayed by enhanced chemiluminescence. The expected band was observed using the Bio-Rad gel imaging system. The relative concentrations of LC3B, P62, mTOR, and PI3K (normalized to β-actin) were detected using the Gel-Pro Analyzer 4.0 software. The results were representative of three independent experiments.
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