The full-length coding sequence of MeSAUR1 was amplified with V2 primer pair (Table 1 ). The PCR product was ligated into the pCold Pros2 vector (TaKaRa, JPN) at the site of Nde I/Sal I, generating pCold Pros2-MeSAUR1. pCold Pros2-MeSAUR1 was introduced into Escherichia coli strain BL21 (DE3) for protein expression. E. coli cells containing pCold Pros2-MeSAUR1 were cultured in LB medium supplied with 100 mg/L Ampicillin at 37°C. When the OD600 of the culture reaches 0.4–0.8, quickly cool the culture to 15°C in ice water, and then 1.0 mM isopropyl β-D -1-thiogalactopyranoside (IPTG) was added and the cultures were incubated at 15°C, 120 rpm for 24 h. The cells were collected and resuspended in the BugBuster® Protein Extraction Reagent (Novagen, GER) and incubated at 30°C for 1 h. The supernatant was collected and purified with Ni-Charged MagBeads (GenScript, United States). pCold Pros2 was expressed and purified as a control in accordance with the above methods. The purified protein was verified by SDS-PAGE and Western Blotting (Sambrook and Russell, 2001 ). Tag Anti-ProS2 (Takara, JPN) and Goat Anti-Mouse IgG/HRP (Boster, United States) were the primary and secondary antibodies used in Western Blot, respectively.
Tag anti pros2
Manufactured by Takara Bio
Sourced in United States, Japan
The Tag Anti-ProS2 is a laboratory equipment product designed for specific applications. It serves a core function, but a detailed unbiased description without interpretation or extrapolation cannot be provided concisely. Further information from the manufacturer would be required to present a factual account of its features and capabilities.
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Lab products found in correlation
4 protocols using tag anti pros2
1
Cloning and Purification of MeSAUR1
2
DNA-Protein Interaction ELISA Protocol
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DNA-protein-interaction enzyme-linked immunosorbent assay was according to the method described by Brand et al. (2010) (link). The MeAGPS1a promoter biotinylated probe was obtained by PCR with Biotin-MeAGPS1a primer pair (Table 1 ). Tag Anti-ProS2 (TaKaRa, JPN) and Goat Anti-Mouse IgG/HRP (Boster, United States) were the primary and secondary antibodies used in DPI-ELISA.
3
DNA Protein Interaction ELISA Protocol
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DPI-ELISA was carried out according to the method described by Brand et al. [19 (link)]. The biotinylated MeAGPS1a promoter probe was obtained by PCR, with Biotin-MeAGPS1a primer pairs and KU50 genomic DNA as template (forward: 5′-Biotin-CAGCTGCCCCTACCGTTAA-3′ and reverse: 5′-Biotin-TAGCAAGTTCAGATTTGGAAAAAACC-3′). The ELISA micro-well plates used Pierce® Streptavidin High Capacity Coated Plates (Thermo Fisher Scientific, Rockford, IL, USA). Tag Anti-ProS2 (TaKaRa, Tokyo, Japan) and Goat Anti-Mouse IgG/HRP (Boster, Wuhan, China) were used as the primary and secondary antibodies in the DPI-ELISA.
4
Overexpression and Purification of MePHD1 Protein
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To get a large amount of MePHD1 protein, the full-length coding sequence of MePHD1 was amplified with P1 primer pairs and then fused to the Nde I/Sal I sites in the pCold Pros2 vector (TaKaRa, Tokyo, Japan), which generated the pCold Pros2-MePHD1. pCold Pros2-MePHD1 was introduced into Escherichia coli strain BL21 (DE3) competent cells for protein expression. The transformed strains were cultured in LB medium supplied with 100 mg/L Ampicillin, at 37 °C, and 250 rpm until the OD600 of the culture reached 0.4–0.8, and the bacterial cell fluid temperature cooled down to 15 °C. The cultures were incubated at 15 °C and 120 rpm, for 24 h, after adding 1.0 mM isopropyl β-d -1-thiogalactopyranoside. The soluble proteins of the cultured cells were extracted and purified by BugBuster® Protein Extraction Reagent (Novagen, Darmstadt, Germany) and Ni-charged MagBeads (GenScript, Jiangsu, China), respectively. pCold Pros2 was expressed and purified as control. The purified protein was verified by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. When performing Western blotting, Tag Anti-ProS2 (TaKaRa, Tokyo, Japan) and Goat Anti-Mouse IgG/HRP (Boster, Wuhan, China) were used as primary and secondary antibodies, respectively.
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