Monoclonal anti–β-actin antibody (AC026, ABclonal Technology, Wuhan, China), polyclonal anti-H3 antibodies (ab1791, Abcam, Cambridge, UK), polyclonal anti-βCBP antibodies (14 (link)), and polyclonal rabbit antibodies against L. migratoria ATF2 (GenBank: OQ360058) and PKCα (GenBank: QNS30453) were prepared in our laboratory using recombinant protein from Escherichia coli to immunize rabbits. Briefly, fragments encoding portions of ATF2 (amino acids 190 to 360 and 490 to 626) or PKCα (amino acids 165 to 315) were amplified and fused with pET-28a (Promega) to produce the recombinant protein in E. coli rosette cells by 0.1 mM isopropyl β-d -thiogalactoside induction. The target protein was purified using a Ni2+-NTA affinity column (GE Healthcare) and served as an antigen to generate rabbit polyclonal antibodies with ABclonal Technology (Wuhan, China). The polyclonal antibodies against pS-ATF2 were generated by GenScript (Nanjing, China). Antibodies against p-Ser/Thr, pS-PKC substrate, and p-Thr were purchased from Abcam and Cell Signaling Technology.
Protocol detail
Production and Characterization of Polyclonal Antibodies
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Actin
Amino acids
Anti antibodies
Antibodies
Antigen
Atf2
Coli e protein
Escherichia coli
Monoclonal antibody
Pkcα
Promega
Protein
Rabbit
Rabbits
Recombinant protein
Corresponding Organization : Institute of Zoology
Other organizations : Capital Normal University
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Variable analysis
independent variables
- Fragments encoding portions of ATF2 (amino acids 190 to 360 and 490 to 626) or PKCα (amino acids 165 to 315) amplified and fused with pET-28a to produce the recombinant protein in E. coli rosette cells by 0.1 mM isopropyl β-d-thiogalactoside induction
- Presence and levels of monoclonal anti–β-actin antibody, polyclonal anti-H3 antibodies, polyclonal anti-βCBP antibodies, polyclonal rabbit antibodies against L. migratoria ATF2 and PKCα, and polyclonal antibodies against pS-ATF2
- Ni2+-NTA affinity column (GE Healthcare) used to purify the target protein which served as an antigen to generate rabbit polyclonal antibodies
- Recombinant protein from Escherichia coli used to immunize rabbits and generate polyclonal antibodies
- Not explicitly mentioned
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