The extracted proteins were separated by electrophoresis with the 10% SDS-PAGE. Gel was run at 80V for 20 min and 120V for 60 min until samples run off the gel and then transferred onto PVDF membranes at 4°C at 80V for 1.5 h. The target protein APP and BACE-1 were measured using the primary antibody of anti-APP (Bioworld, BS6418, 1 : 100) and BACE1 (Proteintech 12807-1-AP, 1 : 100) and then incubated at 4°C overnight. After being washed three times with TBST, corresponding secondary antibody was used at a dilution of 1 : 2000 (bs-40295G, bs40296 G, Bioss, China), followed by visualization with ECL kit (mixed with 1 : 1, PE0010, Solarbio, China). The exposure was completed in dark room with chemiluminescence gel imaging system (C600, Azure Biosystems, USA). The antibody against GAPDH (1 : 2000, TA-08, Zsbio, China) and β-actin (1 : 2000, bs-0061R, Bioss, China) were used as internal controls. Quantitative results were expressed as a ratio of APP to β-actin and ratio of BACE-1 to GAPDH and then compared in each group to measure relative changes.
Pe0010
Manufactured by Solarbio
Sourced in China, United States
The PE0010 is a laboratory equipment that can be used for a variety of purposes. It is a piece of equipment designed to perform specific tasks within a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
R0010, by Solarbio (4 mentions)
Ipvh00010, by Merck Group (3 mentions)
Ecl kit, by Solarbio (2 mentions)
Bs 0061r, by Bioss Antibodies (2 mentions)
Bs 40295g, by Bioss Antibodies (2 mentions)
Bs40296 g, by Bioss Antibodies (2 mentions)
Ta 08, by ZSGB-BIO (2 mentions)
Ab205719, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab6721, by Abcam (2 mentions)
Bs 50549r, by Bioss Antibodies (2 mentions)
Zb 2301, by OriGene (2 mentions)
Gapdh antibody, by Proteintech (2 mentions)
Bl521a, by Biosharp (2 mentions)
Bs6418, by Proteintech (1 mentions)
Mab348, by Merck Group (1 mentions)
Ab32390, by Abcam (1 mentions)
Ab75993, by Abcam (1 mentions)
Ab183612, by Abcam (1 mentions)
0.45 m pvdf membrane, by Merck Group (1 mentions)
21 protocols using pe0010
1
Western Blot Analysis of APP and BACE-1
2
SDS-PAGE Analysis of APP, BACE1, and PKA
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The extracted proteins were separated by electrophoresis with 10% SDS-PAGE. Gel was run at 80 V for 20 min and 120 V for 60 min and then transferred onto PVDF membranes at 4°C at 80 V for 1.5 h. The target proteins APP, BACE1, and PKA were measured by incubating with the primary antibodies against APP (1 : 2000, MAB348, Millipore, USA), BACE1 (1 : 2000, ab183612, Abcam, USA), p-PKA (1 : 2000, ab32390, Abcam, USA), and PKA (1 : 2000, ab75993, Abcam, USA) at 4°C overnight. After washing three times with TBST, the corresponding secondary antibody was used at a dilution of 1 : 2000 (bs-40295G, bs40296G, Bioss, China), followed by visualization with the ECL kit (mixed with 1 : 1, PE0010, Solarbio, China). The exposure was completed in a dark room with the chemiluminescence gel imaging system (C600, Azure Biosystems, USA). The antibodies against GAPDH (1 : 2000, TA-08, Zsbio, China) and β-actin (1 : 2000, bs-0061R, Bioss, China) were used as internal controls. Quantitative results were expressed as a ratio of APP to GAPDH, BACE1 to β-actin, and p-PKA to PKA and then compared in each group to measure relative changes.
3
Western Blot Analysis of Liver Proteins
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Liver tissues collected from the mice were homogenized using a Bioprep-24 homogenizer, and proteins were extracted in RIPA buffer (R0010, Solarbio, Beijing, China) according to the manufacturer’s protocol. Protein concentrations were determined by BCA (Bicinchoninic acid) protein assay kit (PA115, Tiangen, Beijing, China). A 25-μg sample of protein from each group was subjected to SDS-PAGE and transferred to 0.45-µm PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 2 h at room temperature with 5% skim milk in TBST, and the membranes were respectively incubated with primary antibodies against Bax (BS1030), Bcl-2 (BS1511), Caspase 3 (BS1518) (Bioworld Technology Inc., Minneapolis, MN, USA), β-actin (MA5-15739), and NF-κB p65 (PA1-186) (Invitrogen, Carlsbad, CA, USA). Secondary antibody was HRP-conjugated secondary antibody (31430, Invitrogen, Carlsbad, CA, USA) in TBST with skim milk. The signaling proteins were detected using chemiluminescence with the enhanced chemiluminescence (ECL) reagent (PE0010, Solarbio, Beijing, China). Band signal intensities were quantified using ImageJ (NIH Image, Bethesda, MD, USA), and normalized to β-actin.
4
Western Blot Analysis of Apoptosis Markers
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Cells were centrifuged and washed in PBS and resuspended in Tris containing protease inhibitor (50 mM Tris-HCL, 150 mM NaCl). The protein concentration was determined using a Pierce™ BCA Protein Assay Kit (23225, Thermo Fisher Scientific). Then, an appropriate volume of protein was separated on 8%-12% SDS-PAGE and transferred onto PVDF membranes (LM-937D, LMAI Bio, Shanghai, China). The membranes were blocked and washed in 5% nonfat milk for 1 hour, followed by an incubation with the primary antibodies against B-cell lymphoma-2 (Bcl-2, 1 : 1,000, sc-7382, SANTA CRUZ, CA, USA), Bcl-2-associated x, (Bax, 1 : 5,000, sc-7480, SANTA CRUZ), caspase-3 (1 : 1,000, ab208161, Abcam, Inc., Cambridge, MA, USA), cleaved caspase-3 (1 : 500, ab2302, Abcam), β-catenin (1 : 1,000, ab208161, Abcam), cyclin D1 (1 : 500, sc-8396, SANTA CRUZ), and β-actin (1 : 1,000, #3700, Cell Signaling Technology) at 4°C for 16 hours. After that, the membranes were washed and further incubated with secondary antibodies to IgG (1 : 5,000, ab205719, Abcam) and cleaved caspase (1 : 10,000, ab205718, Abcam) at 20°C for 2 hours. The protein bands were developed using enhanced chemiluminescence western blotting substrate (PE0010, Solarbio), and the images were captured and analyzed using a Western Blot Imaging System (FluorChem M, ProteinSimple).
5
Western Blot Analysis of Signaling Proteins
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The extracted proteins were separated by electrophoresis with 10% SDS-PAGE. The gel was run at 80 V for 20 min and 120 V for 60 min and then transferred onto PVDF membranes at 4°C and 80 V for 1.5 h. The target proteins APP, P-JNK1/2, MKK4, MKK7, c-Jun and caspase-3 were measured by incubation with the primary antibodies against APP (1:2000), JNK1/2 (1:1000), P-JNK1/2 (1:500), MKK4 (1:2000), p-MKK4(1:500), MKK7 (1:1000), p-MKK7(1:500), c-Jun (1:1000) and caspase-3 (1:1000) at 4°C overnight. After the gels were washed three times with TBST, the corresponding secondary antibody was used at a dilution of 1:2000, 1:2000, 1:1000, 1:2000, 1:1000, 1:2000, 1:2000, 1:1000 or 1:2000, respectively, followed by visualization with an ECL kit (mixed with 1:1, PE0010, Solarbio, China). The exposure was completed in a dark room with a chemiluminescence gel imaging system (C600, Azure Biosystems, United States). Antibodies against GAPDH (primary antibody 1:5000 and secondary antibody 1:100000) were used as internal controls. Quantitative results are expressed as a ratio of target proteins to GAPDH and then compared to all groups to measure relative changes. Information on the primary antibodies are displayed in Table 2 .
6
Quantifying Renal Ki67 Expression
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The relative levels of Ki67 expression in individual samples were measured by Western blotting. Briefly, the kidney tissues were homogenized and after centrifugation, the protein concentrations in the tissue lysates were determined using a commercial bicinchoninic acid (BCA) kit (ThermoScientific). Subsequently, the tissue lysates (20 μg/lane) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% fat-free dry milk in Tris-buffered saline, 0.1% Tween 20 (TBST) and probed with primary antibodies against Ki67 (1:100, ZSGH-Bio, Beijing, China) or β-actin overnight. The bound antibodies were detected with HRP-coupled goat anti-rabbit IgG (1:3,000 dilution, SE134, Solarbio, China), and visualizing with the enhanced chemiluminescent system (PE0010, Solarbio). The relative levels of Ki67 expression were quantified by densitometric scanning using ImageJ software.
7
Quantitative Protein Expression Analysis
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Cells were added with RIPA lysate (R0020; Solarbio) and protease inhibitor PMSF (P0100; Solarbio) with a final concentration of 1 mM, lysed and centrifuged at 15,000 x g for 10 min at 4˚C to extract the cell protein. The protein concentration was quantified by BCA method, and the final protein concentration was adjusted. The sample protein concentration was 5-10 µg/µl with 10 µl applied to each well, and the gel was electrophoresed using an 8% SDS-PAGE separation gel. After electrophoresis, the protein was transferred to the PVDF membrane by a semi-wet transfer method. 5% Skim milk powder was blocked for 1 h, and then Toll-like receptor (TLR)4 (1:2,000, 66350-1-Ig; Proteintech), TLR7 (1:500, 17232-1-AP; Proteintech), GAPDH (1:5,000, 60004-1-Ig; Proteintech) were added. The primary antibody dilution solution was left at 4˚C overnight, then it was washed three times with 0.1% PBST. The corresponding HRP-labeled goat anti-rabbit (mouse) immune secondary antibody (1:3,000, SA00001-1/2; Proteintech) was added, then incubated at room temperature for 1 h. Then washed 3 times with 0.1% PBST. ECL chemiluminescence solution (PE0010; Solarbio) was added, after that, it was developed, fixed and images were taken in a dark room (LAS 4000; ImageQuant) for recording. ImageJ software was used for image analysis.
8
Western Blot Analysis of Protein Expressions
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Total protein in the cells was extracted using radioimmunoprecipitation assay buffer (R0010, Solarbio, Beijing, China) and quantified using a protein bicinchoninic acid analysis kit (71285-3, Sigma). Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes (3010040001, Millipore, Billerica, MA, USA). After being sealed in 5% skim milk for 120 min at room temperature, the membranes were treated with specific primary antibodies overnight at 4°C and with the secondary antibody for 120 min at room temperature. Protein bands were detected with enhanced chemiluminescence solution (PE0010, Solarbio) and imaged with a GelDoc Go system (Bio-Rad, Hercules, CA, USA). Relative expression of proteins was measured using ImageJ with GAPDH as an internal reference. The antibodies used in the experiments are as follows: primary antibodies were FOXA1 (1 : 1000, ab170933, Abcam, Cambridge, MA, USA), SIX4 (1 : 2000, ab176713, Abcam), GAPDH (1 : 2000, GTX124502, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), p-PI3K (1 : 1000, PA5-118549, Thermo Fisher Scientific), PI3K (1 : 1000, #4257S, Cell Signaling Technologies, Beverly, MA, USA), p-AKT (1 : 1000, ab38449, Abcam), and AKT (1 : 500, ab8805, Abcam); secondary antibody was goat anti-rabbit IgG antibody (ab6721, 1 : 2000, Abcam).
9
APOBEC3 Protein Expression Analysis
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The tissue or SiHa cells were lysed using Biosharp lysis (BL504A). The protein concentration was determined using the bicinchoninic acid assay method. Protein samples (40 µg per lane) were loaded onto a 4% gel, resolved SDS-PAGE and then subsequently transferred onto a PVDF membrane. After blocking with 5% BSA at room temperature for 1 h, the membrane was incubated with anti-APOBEC3G (1:500; bs-15407R; BIOSS), anti-APOBEC3F (1:500; ab227962; Abcam), anti-APOBEC3C (1:500; bs-12495R; BIOSS) and anti-GAPDH (1:5,000; bs-50549R; BIOSS) primary antibodies at 4°C for 16 h. Subsequently, the membranes were incubated with the secondary antibody of goat anti-rabbit HRP conjugated IgG (1:10,000; ZB-2301; OriGene Technologies, Inc.) at 37°C for 1 h in the dark. The blot was developed using the ECL method (PE0010; Beijing Solarbio Science & Technology Co., Ltd.). Protein bands were imaged and analyzed using ImageJ software version 1.52s (National Institutes of Health).
10
Western Blot Analysis of Ago2 Protein
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Protein homogenates were prepared with 5× protein loading buffer (HEART R0891, Xi’an, China) and denatured at 95°C for 5 min. Fifteen micrograms of protein per sample was resolved on a precast 10% (w/v) SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore IPVH00010, Bedford, MA, United States). Blots were blocked with 5% (w/v) nonfat milk solution (in Tris-buffered saline with 0.1% Tween-20 (TBST)) and then incubated overnight at 4°C in primary antibody solutions (Anti-Ago2, Abcam, ab186733, diluted 1:2000). Membranes were then washed with TBST and probed with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000) for 1 h at room temperature. Membranes were visualized using an enhanced chemiluminescence detection kit (Solarbio PE0010, Beijing, China) and quantified with ImageLab 1.46 (BioRad, United States).
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