The cells were washed with cold PBS and lysed in RIPA buffer (P0013B, Beyotime) containing 1% PMSF (ST506, Beyotime). The cell lysates were centrifuged at 12,000 × g for 15 min to remove the debris, and the supernatants were kept for further analysis. The samples were mixed with 25% SDS-PAGE Sample Loading Buffer (P0015; Beyotime) and incubated at 100 °C for 3–5 min. They were then run on an 8–12% SDS-PAG and transferred onto a polyvinylidene fluoride membrane. Subsequently, the membrane was blocked with 2 h of incubation with 5% BSA (SW3015, Solarbio) at room temperature and incubated with the antibodies overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated secondary antibodies were used to detect the primary antibodies. The ECL detection system (WBKLS0100; Millipore, Billerica, MA, USA) was used to visualize the protein bands. The expression values of the proteins analyzed were normalized to the expression of GAPDH. All the experiments were repeated at least three times and yielded consistent results.
P0015
Manufactured by Beyotime
Sourced in China
The P0015 is a laboratory equipment product. It is designed for basic laboratory tasks. The core function of the P0015 is to provide a platform for carrying out various experiments and analyses in a controlled environment.
Automatically generated - may contain errors
Lab products found in correlation
Ipvh00010, by Merck Group (7 mentions)
St506, by Beyotime (3 mentions)
P0013, by Beyotime (3 mentions)
Wbkls0100, by Merck Group (2 mentions)
Iseq00010, by Merck Group (2 mentions)
P0012, by Beyotime (2 mentions)
Ab6721, by Abcam (2 mentions)
P0020, by Beyotime (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
P0018, by Beyotime (2 mentions)
A0216, by Beyotime (2 mentions)
Sw3015, by Solarbio (1 mentions)
Ecl developer, by Thermo Fisher Scientific (1 mentions)
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Bca protein concentration assay kit, by Beyotime (1 mentions)
Sds page electrophoresis solution, by Beyotime (1 mentions)
Trizol lysis buffer, by Thermo Fisher Scientific (1 mentions)
Sds page gel preparation kit, by Solarbio (1 mentions)
30 protocols using p0015
1
Western Blot Protein Expression Analysis
2
Comprehensive Protein Analysis Protocol
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The main reagents used in this study included TRIzol lysis buffer (Invitrogen, USA); reverse transcription kit: PrimeScript RT reagent kit with GDNA Eraser (RR 047A, Takara, Japan); qPCR kit: Tb Green Premix EX TAQ II (RR 820A, Takara); animal whole protein extraction kit (C510003, Sangon Biotech, China); BCA protein concentration assay kit (P0010, Beyotime Biotech, China); 5× loading buffer (P0015, Beyotime Biotech); SDS-PAGE gel preparation kit (P1200, Solar BioBeijing, China); SDS-PAGE electrophoresis solution (P00148, Beyotime Biotech); 10× EMT (D1060, Solar BioBeijing); methanol (CB/T693-1993, Shanghai Guangnuo Chemical Technology Co., Ltd., China); skimmed milk powder (D8340, Solar BioBeijing); 10×TBST buffer (powder) (T1087, Solar BioBeijing); nitrocellulose membrane (NC membrane) (37412133, PALL, USA); and ECL developer (34095, Thermo Fisher, USA).
3
Western Blot Analysis of Hippocampal and Caudate-Putamen Proteins
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The protein blots were prepared according to the conventional procedures of Western blot analysis. Hippocampal and CP tissues were collected from other four rats per group, followed by washing, homogenization, and lysing in RIPA buffer containing protease inhibitors. After conventional protein extraction from the hippocampal and CP tissues, the total protein concentration was measured, followed by adding 30 μg total protein to 1/4 volume of 5 × SDS-PAGE (P0015, Beyotime Biotechnology) sample buffer, boiling the protein sample for 10 min, and separating the proteins by SDS-PAGE. After transferring the separated proteins onto the PVDF membrane (IPVH00010, Millipore), the membrane was incubated with primary antibodies such as doublecortin (1:2,000), nestin (1:2,000), NeuN (1:2,000), ZO-1 (1:2,000), FR-α(1:2,000), RFC(1:2,000), PCFT (1:2,000), GAPDH (1:5,000), and β-actin (1:5,000) overnight at 4°C, and secondary antibodies (1:5,000; Abcam) were incubated for 2 h at 37°C. The absorbance values of protein bands were scanned by using an Odyssey infrared gel imaging system (Affinity, USA). Densitometry was performed to quantify the signal intensity by ImageJ software (version 1.45 J; National Institutes of Health, Bethesda, MD, United States).
4
Protein Extraction and Analysis from Spermatogenic Cells
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The protein lysis buffer (Beyotime, P0013B, China) containing protease inhibitors (Roche, 11,697,498,001, Switzerland) was added to lysate the spermatogenic cells, and incubated on ice for 30 min. The lytic mix liquids were centrifugated at 12,000 × g for 15 min to collect the supernatants. The protein was quantified with BCA method (Beyotime, P0012S, China), then mixed with 5 × loading buffer (Beyotime, P0015, China) and denatured at 95℃ for about 10 min. Subsequently, 15 μg of total protein and 5 μL of protein Marker (P0068, Beyotime) was added to 10% SDS-PAGE electrophoresis gel (Beyotime, P0690, China), respectively. Electrophoresis was performed at 90 V and the voltage was adjusted to 120 V when the Marker ran past the concentrated gel, and then the protein was transferred to PVDF membrane. After blocked with 5% nonfat-dried milk buffer, primary antibody (PLZF/GFRA1/β-actin/SYCP3/DDX4/c-Kit) was added to change the blocking buffer and incubated overnight at 4℃. HRP-labeled secondary antibody was incubated with the membrane at room temperature in an hour, and then the ECL Chemiluminescent substrate (4A BIOTECH, w011-20, China) was added to cover the PVDF membrane. Finally, the image was recorded with chemiluminescence imager (ChemiDoc Touch, Bio-Rad).
5
Western Blot Analysis of HPV18 E7 Protein
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Cells were lysed in RIPA lysis buffer (Beyotime, P0013B) and PMSF (Beyotime, ST506). Cell lysis was carried-out for 30 min on ice, lysates were then centrifuged to remove insoluble material. Then, the loading buffer (Beyotime, P0015) was added to the lysates and heated at 100 °C for 3–5 min to denature the protein completely. Samples were separated on 12% SDS-PAGE Gel (Beyotime, P0053A), transferred to PVDF membranes, and detected with the indicated antibodies. The following antibodies were used for western blotting: anti-HPV18 E7 antibody (8E2, abcam, ab100953, dilution: 1:1000), anti-β-actin antibody (2D4H5, proteintech, 66009-1-Ig, dilution: 1:5000), HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (proteintech, SA00001-1, dilution: 1:2000).
6
SDS-PAGE Analysis of Protein Powder
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The SDS-PAGE experiment was completed according to the method of Grudniewska et al., with some minor modifications [17 ]. Briefly, 1 mg of RPI powder was accurately weighed and dissolved in 1 ml of distilled water. A total of 50 μl of the solution obtained by dissolution was measured and mixed evenly with 200 μl of sample buffer solution (P0015, Beyotime, Shanghai, CHN). Then, the mixture was placed in a boiling water bath for 10 min. An aliquot (20 μl) of protein standard markers and the prepared samples were loaded on to the gel (5% stacking gel and 15% separating gel). The voltage of gel electrophoresis was set to 80 V and Coomassie blue fast staining solution was used to dye the gel in the experiment, and then wash it with distilled water.
7
Immunoblotting of Macrophage Proteins
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The isolated peritoneal macrophages were lysed with 100 μl of 1× cell lysis buffer (9803S; CST), ultrasonicated, mixed with 25 μl of 5× SDS loading buffer (P0015; Beyotime), and finally boiled for 15 min. The resulting samples were subjected to an immunoblotting protocol (43 (link)) for the detection of specific proteins.
8
Protein Expression Analysis via Western Blot
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Erythrocyte‐depleted BM cells were lysed with 1 × sodium dodecyl sulfate (SDS) buffer (P0015, Beyotime, Shanghai, China) and boiled at 100°C for 5 min. The cell extractions were resolved by SDS‐polyacrylamide gel electrophoresis (PAGE) and electrotransferred to polyvinylidene difluoride (PVDF) membranes (ISEQ00010, Sigma, Darmstadt, Germany), followed by overnight incubation with the primary antibodies at 4°C. Horseradish peroxidase‐conjugated anti‐rabbit antibody and anti‐mouse antibody (401353, 401253, Millipore, Burlington, Massachusetts, USA) were used as the secondary antibodies, and Immobilon Western Chemiluminescent HRP Substrate (WBKLS, Sigma) was used for the detection. A comprehensive list of antibodies for WB analysis is included in Supplementary Table S1 .
9
Western Blot Protocol for Protein Analysis
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Prepared cells were dissolved in 100 µL RIPA lysis buffer (CW2333S, CWBIO) including 100× protease inhibitor (CW2200, CWBIO). After centrifugation, the lysates were denatured for 5 min in 5× SDS-PAGE loading buffer (P0015, Beyotime). Proteins were separated on an SDS-PAGE gel and transfered to a PVDF membrane. Next, we block it with 5% skimmed milk powder at 4 °C overnight. After that, the membrane was incubated with primary antibodies for 1 h, then the corresponding secondary antibodies at room temperature for 0.5 h. Developed membranes were imaged using an Azure c300 digital imager system (Azure Biosystems, Dublin, CA).
10
Quantifying Protein Expression in Neurodegenerative Disease
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Protein expression levels of APP, Beclin-1, p62, LC3, Aβ1-42, and β-actin were detected by western blot. After cells were treated for 24 h, they were collected, and total proteins were extracted using RIPA buffer (P0013B, Beyotime) mixed with a protease inhibitor mixture (P1005, Beyotime). Protein concentrations were determined using a BCA assay kit (P0012, Beyotime). Protein samples mixed with loading buffer (P0015, Beyotime) were separated via SDS-PAGE (P0012AC, Beyotime) and transferred to PVDF membranes (FFP24, Beyotime). The membranes were blocked with 5% (w/v) BSA (4240GR025, Biofroxx) for 1 h at room temperature and incubated with primary antibodies (1:1,000 dilution) at 4°C overnight. Subsequently, the membranes were treated with HRP-conjugated goat anti-rabbit antibodies (1:1,000 dilution). Finally, the membranes were visualized with an enhanced chemiluminescence reagent kit (WBKLS0100, Millipore, United States) and photographic imaging equipment (ChemiDoc MP, Bio-Rad, United States). Relative band intensities were quantified using Image J software and normalized to β-actin.
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