The extracted total protein or viral lysates were denatured by adding loading buffer (5 × 250 mM tris-HCl, pH 6.8, 5% dithiothreitol, 10% SDS, 0.5% bromophenol blue, 50% glycerol), followed by boiling the lysates for 5 minutes at 100 °C. Proteins were analysed by SDS-PAGE and then transferred to nitrocellulose membranes using an electroblotter (Bio-Rad Laboratories). The membranes were blocked by 5% non-fat dry milk in TBS-T solution (20 mM Tris [pH 7.4], 150 mM NaCl, 0.1% Tween-20) for 1 hour and then washed three times for 10 minutes each in the TBS-T solution. Membrane samples were probed with a monoclonal antibody specific to the HCV core protein (diluted to 1 μg/mL; Abcam, Ltd.) or Hsc70 (diluted to 0.2 μg/mL; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). As a control, probing experiments were performed using a polyclonal antibody against actin (diluted to 0.2 μg/mL; Santa Cruz Biotechnology, Inc.). After being washed with TBS-T, the membranes were incubated for one hour at room temperature with the appropriate secondary antibody: goat anti-mouse for the HCV core protein, goat anti-rat for Hsc70, and goat anti-rabbit for actin (ZSGB-BIO, China). The protein signal was visualized and captured using Immobilon Western Chemiluminescent HRP Substrate ECL working solution (Millipore Inc.) and a ChemiDoc™ XRS gel imager system (Bio-Rad, CA).
Electroblotter
Manufactured by Bio-Rad
Sourced in United States
The Electroblotter is a laboratory instrument used for the transfer of proteins or nucleic acids from a gel to a membrane support, such as nitrocellulose or PVDF. The Electroblotter applies an electric current to facilitate the transfer process, enabling the efficient and reliable detection of the target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Cytiva (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Hsc70, by Santa Cruz Biotechnology (1 mentions)
Immobilon western chemiluminescent hrp substrate ecl working solution, by Merck Group (1 mentions)
Bca assay, by Sangon (1 mentions)
Imagequant las 4000, by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Bioss Antibodies (1 mentions)
Sds page sample loading buffer, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Tris buffered saline (tbs), by Solarbio (1 mentions)
Alliance mini hd9 system, by Uvitec (1 mentions)
Hrp conjugated goat anti rabbit igg, by Bioss Antibodies (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Hrp conjugated goat anti rabbit or anti mouse secondary antibodies, by Santa Cruz Biotechnology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
5 protocols using electroblotter
1
Western Blotting of HCV Core Protein
2
Protein Expression Analysis in Colon Cancer
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Protein extracts were prepared by lysing colon cancer cells or tumor tissues. Total protein concentrations were determined by BCA assay (Sangon Biotech, China). Aliquots of cell extractswere separated by SDS-PAGE and transferred to a PVDF membrane (Amersham Pharmacia Biotech) by electroblotter (Bio-Rad). Membranes were blocked and incubated overnight with primary antibodies against PARP, cleaved-PARP, total and cleaved-caspase3, total and cleaved-caspase8, total and cleaved-caspase9, total and p-JNK, total and p-p38MAPK (these antibodies used at 1:2000dilution), Bid, Bad, Bax, Bak, t-STAT3 (1:1000dilution), p-STAT3, t-STAT5, p-STAT5 (these antibodies used at 1:1000 dilution), and β-actin(1:2000dilution) at 4°C overnight. Then the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse IgG (1:3000 dilution) for one hour at RT. All these antibodies were purchased from Cell Signaling Technology Inc. Membranes were subsequently incubated with ECL kits (Pierce/Thermo Fisher Scientific) and developed on chemiluminescence imaging system (ImageQuant™ LAS 4000).
3
Small RNA Isolation and Northern Blotting
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RNA blotting studies were done as reported earlier [21 (link)]. Briefly, from the total RNA a small fraction of RNA was isolated using a miR isolation kit (Thermo Fisher USA). About 5 µg of the RNA fraction was subjected to separation on a polyacrylamide gel (10%) by electrophoresis, then the resultant was transferred to a hybridization membrane using an electroblotter (Bio-Rad USA) at 4°C for 20 min. The RNAs were cross linked using 0.1 M each of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 1-methylimidazole for 2 h at 55°C at pH 8.4.
4
Western Blot Analysis of Nanog Protein
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The monoclonal anti-Nanog antibody from medaka was provided by Professor Hongyan Xu [36 (link)]. The total protein of tissues was extracted with RIPA Lysis Buffer (Beyotime, Shanghai, China) and mixed with SDS-PAGE Sample Loading Buffer (Beyotime). After being boiled for 5 min, 10 µL protein buffer was loaded into a lane, electrophoresed through 10% SDS-PAGE gels, and electroblotted onto polyvinylidene difluoride membrane (Merck Millipore, Billerica, MA, USA) by an electroblotter (BioRad, Hercules, CA, USA). The membrane was blocked with 5% BSA (Solarbio) for 1 h. After being washed with TBS (Solarbio), the membrane was incubated with anti-β-Actin antibody (Bioss, Beijing, China) or anti-Nanog antibody (1:1000 dilution in TBS) at 4 °C overnight. Then, the membrane was washed with TBS and incubated with HRP-conjugated goat anti-rabbit IgG (Bioss) (1:2000 dilution in TBS) for 2 h. Finally, protein blots were colored with Chemiluminescent Substrate for Western blotting Kit (Cyanagen, Bologna, Italy) and imaged by an Alliance MINI HD9 system (Uvitec, Cambridge, UK). The β-Actin was used as an internal control.
5
Western Blot Analysis of Stat6 Activation
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Protein extracts were prepared by lysing the macrophages for 5 min in boiling denaturing extraction buffer containing 1% SDS and 10 mM Tris (pH 7.4). The insoluble material was removed by centrifugation for 15 min at 12 000g at 4 °C. The total protein concentrations were determined using the BCA assay (Pierce/Thermo Fisher Scientific, Rockford, IL, USA). The cell extracts (standardized to 50 µg of total protein/lane) were separated by 12% denaturing SDS–PAGE and transferred to a PVDF membrane (Amersham Pharmacia Biotech, Freiburg, Germany) by semidry blotting using an electroblotter (Bio-Rad, Hercules, CA, USA) at 0.8 mA/cm2 for 120 min. The membranes were blocked by 5% milk with PBST and incubated overnight with primary antibodies against total-Stat6, phospho-Stat6 (pSTAT6-Tyr641) and β-Actin (all from Cell Signaling Technology, Beverly, MA, USA) at 4 °C. After being washed in PBST three times, the membranes were incubated for 45 minutes with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in blocking buffer. The membranes were subsequently washed and incubated with ECL kits (Pierce/Thermo Fisher Scientific, Rockford, IL, USA). The signal intensity was quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA). The western blotting experiment was repeated at least three times.
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