Pierce his protein interaction pull down kit

Y2H assays were performed using a Y2H Gold Yeast system (Clontech, CA, USA) [69 (link), 83 ]. The transformed yeast cells were grown on a selective medium (SD/−T/−L) (Takara, Dalian, China), and yeast cells cultured for 3 days were transferred to SD/−T/−L/−H/−A medium (Takara) for another 3 days.
Pull-down assays were performed using a Pierce™ His Protein Interaction Pull-down Kit (ThermoFisher Scientific, Waltham, MA, USA) [69 (link), 83 ]. The recombinant plasmids were transformed into Escherichia coli BL21 cells (TransGen Biotech, Beijing, China) and the fusion proteins were obtained by IPTG induction. The corresponding fusion protein combinations were incubated in nickel affinity chromatography. The eluent was tested with anti-GST and anti-HIS antibodies (Abmart, Shanghai, China).
For the BiFC assay, Agrobacterium solution containing recombinant plasmids was injected into tobacco leaves. The transformed tobacco plants were cultured under light conditions for 2 days. The fluorescence signals were observed using confocal microscopy.

His-CASP10^mut was bacterially expressed and purified. Recombinant ACLY protein was procured from Sino Biological Inc. In vitro binding assay was then performed using Pierce His Protein Interaction Pull-Down Kit (ThermoFisher Scientific) following the manufacturer’s protocol.

Protein binding partners of DNAJA1-107 were identified using the Pierce™ His Protein Interaction Pull-Down Kit from ThermoFisher Scientific. 6x-His tagged DNAJA1-107 was expressed in One Shot™ BL21 (DE3) chemically competent E. coli and immobilized on a cobalt resin. A negative control was performed by incubating the cell lysate with only the cobalt resin to identify non-specific binding. In Gel Digestion was performed as previously described [51 (link)]. Trypsin-digested samples were analyzed by LC-MS using an Acquity UPLC M-Class and a Xevo G2-XS Quadrupole Time-of-Flight Mass Spectrometer with a 60-minute linear gradient from 100% H₂O + 0.1% FA to 100% ACN + 0.1% FA using a 0.35 µL/min flow rate. The MS source was a nanoflow ESI with MSE continuum acquisition. Scan time was 0.5 s with a mass range of 50–2000 Da. ProteinLynx Global SERVER™ by Waters was used for data processing. Peptide fragments were matched to proteins using reverse entry searching in the UniProt database [52 (link)] using FASTA sequences.

The His pull-down assay was performed using the Pierce His Protein Interaction Pull-Down Kit (Thermo-Fisher Scientific, Waltham, MA, USA). Briefly, His-CbpA served as bait and was incubated with HisPur Cobalt Resin at 4 °C for at least 30 min, the resin was washed with wash solution, and incubated with HT-29 cell lysates overnight at 4 °C. HT-29 cell lysates were prepared according to the user guide of the Pierce His Protein Interaction Pull-Down Kit. Briefly, HT-29 cells were released from the surface of the flask by Lifters, and harvested by centrifugation (500× g, 5 minutes), then washed once with 1 mL of Tris Buffered Saline (TBS) per 5 mL of original cell culture volume. HT-29 cell pellet was resuspended with 2.5 mL of ice-cold TBS per gram wet weight of cells, and added 5 mL of Pierce Lysis Buffer per gram wet weight of cells, and then incubated on ice for ~30 minutes. Supernatant was obtained by centrifuging at 12,000× g for 5 minutes. Finally, the resin was washed with elution buffer, and the elution buffer containing bait and prey proteins was analyzed using SDS-PAGE.

We introduced point mutations in the FaeG proteins using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies, USA). His-tagged APN proteins were expressed at 16 °C and loaded on a Pierce™ His protein interaction Pull-down kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions [24 (link)]. We performed SDS-PAGE and western blotting to determine the interaction between FaeG point mutants and APN. We incubated the blots overnight in a monoclonal antibody against F4⁺ or polyclonal antibodies against APN, and stained these using enhanced chemiluminescence (ECL) (Pierce, USA) reagents.