The constructs of pGEX-G4S-rFc and pAG-MNase were transformed into E. coli BL21, and the GST-rFc and protein A-protein G-MNase-His fusion proteins were induced at 37°C for 3 h using 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). In vitro pull-down assays were performed using a Pierce Pull-Down PolyHis Protein:Protein Interaction kit (Thermo Fisher Scientific 21277) and analyzed by Western blotting with 6xHis-tag antibody (Invitrogen MA1-21315) and GST tag antibody (Invitrogen MA4-004).
6xhis tag antibody
Manufactured by Thermo Fisher Scientific
The 6xHis-tag antibody is a laboratory tool used to detect and purify proteins that have been engineered to contain a 6xHis-tag sequence. The 6xHis-tag is a short peptide that can be attached to the target protein, allowing for specific recognition and isolation of the protein using this antibody.
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2 protocols using 6xhis tag antibody
1
In vitro Protein-Protein Interaction
2
Thermal Denaturation Assay for ClpP
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ClpP proteins were expressed using the same method, and the supernatant of the lysate was collected by centrifugation after ultrasonic fragmentation. Nepetin at 200 μM and an equal volume of DMSO were incubated with the supernatant at room temperature for 1 h, after which the insoluble precipitate was removed by centrifugation again (20,000 g, 20 min, 4°C). The resulting solution was aliquoted into PCR tubes and heated for 5 min at the indicated temperature in a Mastercycler® nexus gradient (Eppendorf) temperature cycler, followed by immediate immersion in an ice bath for 3 min. The samples were centrifuged to remove insoluble material and taken for western blot analysis. 6x-His Tag Antibody (Cat# MA1– 21315, Invitrogen) was used to detect ClpP protein, and the relative intensities of the target proteins were analyzed using ImageJ software.
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