Mouse anti his antibody

EBY100/pYD1-ORF25 pellets were incubated with 1:1000 diluted mouse anti-His antibody (ABclonal, China) for 1 h. After washing three times with PBS, Cy3-conjugated AffiniPure goat anti-mouse IgG (Invitrogen, USA) at a dilution of 1:500 was added and incubate at RT for 40 min, then washed three times and resuspended in 500 μl of sterile PBS. Stained bacteria were cytospinned on glass slides and mounted with fluorescent microscopy mounting solution. Images were captured using an Olympus BX53 fluorescence microscope and analyzed with the iVision-Mac scientific imaging processing software (Olympus, Japan).

Purified recombinant proteins were mixed with 5 × SDS reducing loading buffer and boiled for 8 min at 100 °C and then resolved on 10% to 12% SDS-PAGE followed by Coomassie brilliant blue staining. For Western blot analysis, purified recombinant proteins, protein pulldowns, and eluted proteins were separated by 10% to 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore) and block with 5% skimmed milk in Tris-buffered saline-Tween 20 for 2 h at room temperature (RT). After washing with Tris-buffered saline-Tween 20 three times, the polyvinylidene difluoride membranes were incubated with primary antibodies overnight at 4 °C and then with a horseradish peroxidase-labeled goat anti-mouse or anti-rabbit IgG antibody (SouthernBiotech) for 1.5 h at RT. The fluorescence signals were visualized using an enhanced chemiluminescence kit (New cell & Molecular Biotech) and analyzed using a ChemiDoc MP imaging system (Bio-Rad). The following primary antibodies were used: mouse anti-His antibody (ABclonal), mouse anti-HA antibody (ABclonal), rabbit anti-GST antibody (Proteintech), rabbit anti-Flag antibody (Proteintech), mouse anti-PfGAMA-Tr3 antisera, rabbit anti-ANK1-F2 antisera, and rabbit anti-band 3-P5 antisera.

Cells were fixed with 4% paraformaldehyde (Beijing Dingguo changsheng Biotechnology Co.,Ltd, AR‐0211) in PBS at room temperature for 10 min. Cells were then washed with PBS three times and permeabilized with 0.1% TritonX‐100 at room temperature for 10 min. Cells were washed again three times with PBS and blocked for 3 h with 3% (w/v) BSA in TBST at room temperature. Cells were then incubated with primary antibodies (Rabbit Flag antibody, Sigma‐Aldrich, F7425, 20 µg; Mouse anti‐His antibody, ABclonal, AE003 1:200 dilution) in 3% (w/v) BSA for 1 h at room temperature. After washing five times with PBS, cells were incubated with secondary antibodies (Alexa Fluor 488 anti‐rabbit IgG, Cell Signaling Technology, 4412S, 1:2 000 dilution; Alexa Fluor 594 anti‐mouse IgG, Invitrogen, A‐11004, 1:1 000 dilution) in 5% (w/v) BSA for 1 h. After washing five times with PBS, cells were incubated with 1 µg mL⁻¹ DAPI (Solarbio, C0060) for 15 min. Cells were then washed five times and imaged.
The cells were imaged immediately by confocal laser scanning microscope (CLSM, PerkinElmer UltraVIEW VoX, Germany). For DAPI imaging, the excitation was 405 nm, and the emission filter was 447 nm (w60); for Alexa Fluor 488 imaging, the excitation was 488 nm, and the emission filter was 525 nm (w50). for Alexa Fluor 561 imaging, the excitation was 561 nm, and the emission filter was 600 nm (w52).