S. islandicus Cmr-2α-His strain was cultured in SCVy medium. When the absorbance at 600 nm of the culture reached 0.4, cell mass was collected by centrifugation, re-suspended in 50 mM phosphate buffer and sonicated. Then, crude protein samples were loaded on 12% SDS-PAGE and fractionated according to their sizes. Fractionated proteins were transferred onto a nylon membrane using the Semi-Dry Electrophoretic Transfer Cell system (Bio-Rad; Hercules, CA, USA). The membrane was incubated with a hybridization buffer containing an antiserum against His-tag peptide (GenScript, Piscataway, NJ, USA) during which the antiserum bound to His-tagged Cmr-2α protein. The His-tag antiserum was then recognized by a secondary antibody (Goat Anti-Mouse IgG, GenScript) and the protein bands were visualized by chemiluminescent detection using the clarity Western ECL substrate (Bio-Rad; Hercules, CA, USA) and recorded using the MFChemibis 3.2 imaging device (DNR; Jerusalem, Israel).
Semi dry electrophoretic transfer cell system
Manufactured by Bio-Rad
Sourced in United States
The Semi-Dry Electrophoretic Transfer Cell system is a lab equipment designed for the transfer of proteins or nucleic acids from polyacrylamide or agarose gels to a membrane support, such as nitrocellulose or PVDF. The system facilitates the efficient and uniform transfer of molecules from the gel to the membrane for further analysis or detection.
Automatically generated - may contain errors
Lab products found in correlation
Clarity western ecl substrate, by Bio-Rad (4 mentions)
Goat anti mouse igg, by GenScript (2 mentions)
Horseradish peroxidase labeled goat anti rabbit antibody, by Beyotime (2 mentions)
Amersham imager 600 device, by GE Healthcare (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Ecl western blot substrate, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose blotting membrane, by GE Healthcare (1 mentions)
Coomassie protein assay kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
6 protocols using semi dry electrophoretic transfer cell system
1
Purification and Detection of His-tagged Cmr-2α Protein
2
Protein Expression and Western Blot Analysis
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Sulfolobus transformants were cultured in ACVy medium to induce expression of csa3a gene under control of araS promoter. When OD600 reached 0.2, cells were harvested and sonicated. Crude protein samples were separated by 12% sodium dodecyl sulphate (SDS)-PAGE, and then transferred to a nylon membrane using the Semi-Dry Electrophoretic Transfer Cell system (Bio-Rad; Hercules, CA, USA). The target proteins were detected by rabbit polyclonal antibodies against Csa1, Cas1 and Csa3a, and then bound by horseradish peroxidase-labeled goat anti-rabbit antibody (Beyotime, Beijing, China). Bands were visualized by chemiluminescent detection using the clarity Western ECL substrate (Bio-Rad; Hercules, CA, USA) and the MF-Chemibis 3.2 imaging device (DNR; Jerusalem, Israel).
3
Western Blot Analysis of His-Tagged Proteins
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Z. mobilis His-ZMO0038 and His-ZMO0038M strains were cultured in RMG2 medium. Cells were then harvested by centrifugation when OD600 nm reached 0.4, followed by suspension in 50 mM phosphate buffer for sonication lysis. The crude protein samples were fractionated on 12% SDS-PAGE and transferred onto a nylon membrane using the Semi-Dry Electrophoretic Transfer Cell System (Bio-Rad, Hercules, CA, USA). The membrane was incubated with a hybridization buffer including an antibody against His-tag peptide (GenScript, Nanjing, China). The antibody bound to the His-tagged proteins was then recognized by a secondary antibody (Goat Anti-Mouse IgG, GenScript, Nanjing, China). The results were then visualized by chemiluminescent detection using the clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) and analyzed using the Amersham Imager 600 device (GE Healthcare, Seoul, South Korea).
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Z. mobilis His-ZMO0038 and His-ZMO0038M strains were cultured in RMG2 medium. Cells were then harvested by centrifugation when OD600 nm reached 0.4, followed by suspension in 50 mM phosphate buffer for sonication lysis. The crude protein samples were fractionated on 12% SDS-PAGE and transferred onto a nylon membrane using the Semi-Dry Electrophoretic Transfer Cell System (Bio-Rad, Hercules, CA, USA). The membrane was incubated with a hybridization buffer including an antibody against His-tag peptide (GenScript, Nanjing, China). The antibody bound to the His-tagged proteins was then recognized by a secondary antibody (Goat Anti-Mouse IgG, GenScript, Nanjing, China). The results were then visualized by chemiluminescent detection using the clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) and analyzed using the Amersham Imager 600 device (GE Healthcare, Seoul, South Korea).
4
Protein Extraction and Western Blot Analysis of Sulfolobus Cells
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Cells in 15 ml culture were collected by centrifugation. Cell pellets were re-suspended in 1 ml 10 mM Tris–HCl buffer (pH 8.0). The resulting cell suspensions were sonicated to disrupt Sulfolobus cells. Cell debris was removed by centrifugation 13000 rpm at 4°C for 30 min, yielding cellular extracts for further analysis. Protein concentrations of the samples were determined using Coomassie Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Similar amounts of protein (ca. 10 μg) were taken from the prepared cell extracts and loaded on a 12% polyacrylamide gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, fractionated proteins were transferred from the polyacrylamide gel onto a nitrocellulose blotting membrane (GE Healthcare, Waukesha, WI, USA), using the Semi-Dry Electrophoretic Transfer Cell system (Bio-Rad, Hercules, CA, USA) and used for immunoblotting. Briefly, the membrane was incubated in 5% skim milk blocking agent for 1 h, and then incubated with individual primary rabbit antibodies (against Orc1-2 or PCNA3 proteins) and finally with the horseradish peroxidase-labeled goat anti-rabbit antibody (Beyotime, Beijing, China) as described previously (31 ). Protein bands were visualized using the ECL western blot substrate (Thermo Scientific, Waltham, MA, USA) and recorded by exposure to an X-ray film.
5
Csm3-His Tagged Protein Purification
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The Csm3‐His‐tagged strain was cultured in the TB medium. When the OD600 of the culture reached 2.0, cells (20 ml) were collected by centrifugation. Phosphate buffer (50 mM) was used to resuspend the cell pellets. Afterward, the resulting bacterial suspension was lysed by sonication. Then, the mixture was centrifuged (13,000 rpm, 20°C, 30 min), and the supernatant was fractionated through 12% SDS‐PAGE. Fractionated proteins were transferred onto a PVDF membrane using the Semi‐Dry Electrophoretic Transfer Cell system (Bio‐Rad). Antibody incubation and visualization were performed as previously reported
29 (link).
29 (link).
6
Biotin-Labeled EMSA Probe Preparation
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Electrophoretic mobility shift assay (EMSA) probes were generated by PCR or annealing using the oligonucleotides with one of the primer pair 5′-end biotin-labeled (Supplementary Table S1). Then the products were purified from 6% native polyacrylamide gel electrophoresis (PAGE). The EMSA binding reactions (20 μl) containing 10 pmol of biotin-labeled probes and different concentrations of Csa3a, as described in the figure legends, were incubated for 20 min at 40°C in the binding buffer (20 mM Tris–HCl, pH8.0, 50 mM KCl, 5% glycerol, 1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 5 ng/μl poly(dI-dC)). For specific competition, increasing amounts of unlabeled specific probes were added to the reaction mixture. After the reaction, samples were loaded onto a 6% native PAGE gel buffered with 1× TBE solution. DNA–protein complexes were separated at 100 V for 90 min and then transferred to a polyvinylidenefluoride membrane (Bio-Rad, Hercules, CA, USA) using the Semi-Dry Electrophoretic Transfer Cell system (Bio-Rad). Bands were visualized by chemiluminescent detection using the clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) and the MF-Chemibis 3.2 imaging device (DNR; Jerusalem, Israel).
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