The total proteins of PSCs were extracted using a BBprExtra total protein extraction kit (Bestbio, Shanghai, China). The extracted proteins, along with rEgANXBs and cyst fluid, were separated using 12% SDS-PAGE and transferred to Nitrocellulose Membranes (Biosharp, Hefei, China). Then, the membrane was blocked using 5% skim milk for 2 h. Sheep negative or positive serum diluted 1:100 in phosphate-buffered saline (PBS), rat negative serum, or anti-rEgANXBs rat IgG (dilution 1:400 in PBS) was added and incubated overnight at 4 °C. After washing, horseradish peroxidase (HRP)-conjugated rabbit anti-sheep IgG (dilution 1:1000 in PBS) or HRP-conjugated goat anti-rat IgG (H + L) (ABclonal, Wuhan, China) (dilution 1:2000 in PBS) was added for 2 h incubation, and the immunoreactive signals were detected using a Metal Enhanced DAB Substrate Kit (Solarbio, Beijing, China).
Nitrocellulose membrane
Manufactured by Biosharp
Sourced in China
Nitrocellulose membranes are thin, porous sheets commonly used in various laboratory techniques. They are made from a type of cellulose derived from cotton or wood pulp. Nitrocellulose membranes are designed to allow the transfer and immobilization of biomolecules, such as proteins and nucleic acids, for further analysis and detection.
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Lab products found in correlation
Supersignal west femto substrate trial kit, by Thermo Fisher Scientific (2 mentions)
Metal enhanced dab substrate kit, by Solarbio (1 mentions)
Total protein extraction kit, by Beyotime (1 mentions)
Enhanced ecl reagent, by Biosharp (1 mentions)
Ai600 system, by GE Healthcare (1 mentions)
Bca kit, by Beyotime (1 mentions)
Bromophenol blue, by Bio-Rad (1 mentions)
Tgf βri, by Abcam (1 mentions)
Varioskan flash, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Abcam (1 mentions)
Mammalian protein extraction reagent, by CWBIO (1 mentions)
Enhanced bca protein assay kit, by Solarbio (1 mentions)
Ecl reagent, by Biosharp (1 mentions)
Ripa cell lysis buffer, by Solarbio (1 mentions)
Rabbit anti e cadherin, by Cell Signaling Technology (1 mentions)
Rabbit anti snail, by Cell Signaling Technology (1 mentions)
Mouse anti β actin, by Proteintech (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Defatted milk, by BD (1 mentions)
13 protocols using nitrocellulose membrane
1
Immunoblot Analysis of rEgANXBs in Hydatid Cyst Fluid
2
Liver Protein Extraction and Western Blot Analysis
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Extraction of total protein from liver tissue samples was conducted via the Total Protein Extraction Kit (Beyotime, China); then, the protein concentration was detected using a BCA Kit (Beyotime, China). An equal amount of protein (30 μg) was separated via 12% SOD-PAGE and electroblotted onto nitrocellulose membranes (Biosharp, China). Then, the membranes were blocked with 5% nonfat milk for 2 h and incubated with both anti-Bcl-2 and anti-Bax primary antibodies (1:500) at 4 °C overnight. After washing with TBST, the blot was incubated with HRP-conjugated anti-IgG for 2 h and detected using an enhanced ECL reagent (Biosharp, China) on an AI600 System (GE Healthcare, UK). Relative protein expression was quantified via ImageJ software and normalized against the β-Actin control (1:2000, Sangon Biotech, China).
3
Western Blot Analysis of Embryonic Proteins
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Whole cell lysates from 500 embryos were lysed in 2× Laemmli buffer (0.5 M Tris HCl, pH 6.8, 0.4% SDS, 2% glycerol, 0.5% β-mercaptoethanol) and Bromophenol Blue (Bio-Rad, USA). The lysates were heated at 100 °C for 10 min, then cooled rapidly for 1 min. Proteins were separated by using SDS polyacrylamide gel electrophoresis (90 V for 30 min, 110 V 100 min,) and blotted onto nitrocellulose membranes (Biosharp, China). The membranes were blocked in TBST (TBS containing 0.1% Tween 20) containing 5% non-fat milk at room temperature 1 h, followed by overnight incubation with antibody at 4°C. After being probed with the primary antibodies, the membranes were washed in TBST, incubated with an HRP-linked secondary antibody for 1 h at 37°C, and washed three times with TBST. The bound antibodies were detected with SuperSignal West Femto Substrate Trial Kit (Thermo). The intensity of the protein bands was calculated by ImageJ software (NIH, Bethesda, USA).
4
Western Blot Analysis of Erythrocyte Proteins
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Erythrocytes were lysed on ice using Mammalian Protein Extraction Reagent (CWBiotech, Beijing, China), and protein concentrations were determined using Varioskan Flash (Thermo, Waltham, MA, USA). Lysate supernatants were boiled for 10 min, separated on an 10% SDS-polyacrylamide gel, and then transferred to nitrocellulose membranes (Bio-sharp, Beijing, China). Membranes were blocked for 1 h in 5% skim milk in Tris-buffered saline with Tween (TBST) and were then incubated with antibodies against Band 3 (1:1000, Abcam, Cambridge, MA, USA), TGF-β RI (1:1000, Abcam, Cambridge, MA, USA), Act RII (1:1000, Abcam, Cambridge, MA, USA) and α-tubulin (1:2000, Abcam, Cambridge, MA, USA) overnight at 4 °C. Membranes were washed three times with TBST. Membranes were then incubated with peroxidase-conjugated immunoglobulin G antibody (1:5000; Jackson Immunoresearch, Lancaster, PA, USA) for 2 h at RT before washing with TBST. After adding developing liquid, chemiluminescent signals were detected using a Tanon-5200 system (YuanPingHao Biotech, Beijing, China).
5
Protein Isolation and Western Blot Analysis
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Total proteins were isolated using cell RIPA lysis buffer (Solarbio, Beijing, China, R0020). The extracted protein concentration was determined with an enhanced BCA protein assay kit (Solarbio, Beijing, China, PC0020). Afterward, samples containing 20 μg of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Shanghai Yase Biomedical Technology Co., LTD, Shanghai, China, PG212), and the separated proteins were transferred to nitrocellulose membranes (Biosharp, Beijing, China, ISE00010). The membranes were blocked with blocking buffer (Shanghai Yase Biomedical Technology Co., LTD, Shanghai, China, PS108P) for 1 h. In addition, the membranes were incubated with antibodies against BAX, Bcl-2, GAPDH, and Tublin (β-tublin) (shown in Table 2 ) overnight at 4 °C. The membranes were coincubated with suitable secondary antibodies for an additional 1 h at room temperature. The bands were visualized with enhanced chemiluminescence (ECL) reagent (Biosharp, Beijing, China, BL520B).
6
Protein Extraction and Western Blot Analysis
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Using protein extraction buffer (Solarbio, RIPA lysis buffer, Beijing, China), the protein was extracted from HCC tissues (tumor and matched normal adjacent tissues) and cell lines. Protein concentrations were determined via BCA Protein Assay Kits (Elabscience, Wuhan, China). On a 10% SDS-PAGE, protein sample fractions were separated and the remaining fractions were then transferred to nitrocellulose membranes (Biosharp, Shanghai, China). After being blocked overnight at 4 °C with 5% fat-free milk, primary antibodies were exposed to membranes. After that, secondary antibodies that were HRP-conjugated were applied to the membranes for an hour at room temperature. Mouse anti-β-actin (Proteintech, 66009-1-1g, WB 1:5000, Wuhan, China), mouse anti-NOX4 (Proteintech, 67681-1-1g, WB 1:2000), rabbit anti-E-cadherin, and rabbit anti-Snail (Cell Signaling Technology, both WB 1:1000, Boston, MA, USA) were used as the main antibodies. HRP-conjugated goat anti-rabbit antibodies were used as the secondary antibodies (Cell Signaling Technology, WB 1:5000, Boston, MA, USA).
7
Quantitative Multistrip Western Blotting of Oocyte and Embryonic Proteins
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Western blotting was performed using Multistrip Western blotting protocols described previously 67 . Briefly, whole cell lysates from 500–1,500 oocytes or reconstructed embryos were lysed in beta‐mercaptoethanol‐containing loading buffer and heated at 95°C for 8 min. The embryonic lysates were separated by SDS–PAGE (90 V for 30 min, 30–50 V overnight, when the loading buffer is no longer on the gel, stop the electrophoresis). Following electrophoresis, the protein transferred from the gel to nitrocellulose membranes (Bio‐sharp, China). We next cut the membrane into two strips and blot separately. The membrane was followed by blocking in TBST containing 5% defatted milk (BD, USA) for 120 min at 4°C. After being probed with primary antibodies (overnight at 4°C), the membranes were washed in TBST, incubated with an HRP‐linked secondary antibody for 120 min at 37°C, and washed three times with TBST. Bound antibodies were detected with SuperSignal West Femto Substrate Trial Kit (Thermo, USA). The antibodies used are listed in IF section. Quantification analysis of band intensity was calculated by ImageJ software (NIH, Bethesda, USA, http://rsbweb.nih.gov/ij/ ).
8
Detecting Protein Levels in HCC Cells
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The protein levels in HCC cells were detected by western blot assays, and primary antibodies were used for this assay, including anti-N-cadherin (ab76011) and anti-E-cadherin (ab40772) (1/10,000, Abcam, USA). The cells were collected and lysed in RIPA buffer (Beyotime Biotech). The BCA Protein Assay kit (Beyotime Biotech) was used to measure protein concentrations [29 (link)]. Proteins were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes (Biosharp, Anhui, China) after electrophoretic resolution. Subsequently, the membranes were sealed in 5% nonfat milk powder with TBS and 0.01% Tween and incubated with primary antibodies at 4°C overnight. Next, the membrane was incubated with secondary antibody for 1 h. Finally, an enhanced chemiluminescence kit (Beyotime Biotech) was used to visualize protein bands.
9
Quantification of 5hmC and 5mC in DNA
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Each DNA sample was diluted to 150 ng/µL and denatured at 99°C for 5 minutes. The denatured DNA (2 µL) was spotted on a nitrocellulose membrane (Biosharp, Hefei, China) and air dried. The membrane was blocked by soaking in 5% BSA in TBS-T at room temperature for 1 hour, following which it was immunoblotted with antibody against 5hmC (1:10,000; Active Motif, Carlsbad, CA, USA) and 5mC (1:2,000; Active Motif) at 4°C overnight. Later, the membrane was incubated with horse radish peroxidase-conjugated anti-rabbit IgG (1:40,000 for 5hmC, 1:2,000 for 5mC; Biosharp) at room temperature for 30 minutes. The membrane was then incubated with enhanced chemiluminescent substrate (Biosharp) for 1 minute. Images were taken using the ChemiDocTM Touch Imaging System (Bio-Rad, Hercules, CA, USA).
10
Quantitative Western Blot Analysis
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Western blotting was performed as previously described (15 (link)). In brief, the extracted proteins were transferred onto a nitrocellulose membrane (Biosharp, China), which were blocked with 5% skim milk in tris-buffered saline with tween 20 (TBST) at 4°C overnight, followed by incubation with the primary antibody of SHVV protein (1:1,000) or β-actin (1:1,000) for 2 h at room temperature. The membranes were then washed three times with TBST and then incubated with IRDye 800CW conjugated donkey anti-rabbit antibody (1:10,000) for 1 h at room temperature. The signal intensity was then determined using Odyssey CLx (LI-COR, USA).
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