Ha tag monoclonal antibody

Western blot analysis was performed as described in Wang et al. (2011) (link). The total protein was extracted by using the Plant Protein Extraction Kit (CWBIO, China) from 20-days-old seedlings grown in NP conditions. The total protein (60 μg) was separated on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose filter membrane (Amersham, USA). The membrane was blocked with 5% milk in phosphate-buffered saline. The primary antibody [HA Tag monoclonal antibody (1:5000, Thermo Fisher, United States)] and the secondary antibody [goat anti-mouse IgG-HRP (1:3000, Sangon, China)] were used for the Western blot. The blotted membrane was incubated with ECL luminous solution MaxiLumin-WB (Biokits tech Inc., China) and visualized using an Odyssey FC imaging system (LI-COR, United States).

Western blotting was performed as described by Wang et al. (2011) (link). The primary antibody [HA Tag monoclonal antibody (1:5000, Thermo Fisher, United States)], and the secondary antibody [goat anti-mouse IgG(H+L)-HRP (1:5000, Beijing Protein Innovation, China)] was used for the Western blot. The blotted membrane was detected using an Odyssey FC imaging system (LI-COR, United States).

Protein extracts were obtained from whole cell extracts of the IV.1, MRR1^HA, TAC1b^HA, and UPC2^HA strains after precipitation with trichloroacetic acid as previously described (33 (link)). Proteins were separated through electrophoresis using Mini-PROTEAN TGX gel (Bio-Rad Laboratories, Hercules, CA) and blotted onto nitrocellulose membranes. Mrr1p, Tac1bp, and Upc2p were detected by primary HA Tag Monoclonal Antibody (Invitrogen, ThermoFisher Scientific, Waltham, MA) with 1/2,500 dilution in 5% bovine serum albumin in phosphate-buffered saline with 1% Tween (BSA-PBS-T) and secondary Goat anti-Mouse IgG (H + L) antibody (Invitrogen, ThermoFisher Scientific, Waltham, MA) with 1/500 dilution in BSA-PBS-T.

Protein lysates were prepared and quantified by using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA, #23225). NuPAGE™ 4–12% Bis-Tris Protein Gels and the respective running buffer (Thermo Fisher Scientific; Waltham, MA, USA, #NP0321PK2, #NP0001) were used for separation of 20 μg of each protein sample. Blotted membranes were blocked with 5% milk for 1 h, incubated overnight at 4 °C with the primary antibodies (HA tag monoclonal antibody (26183, Invitrogen, 1:5,000), anti-CYBA antibody (sc-271968, Santa Cruz, Heidelberg, Germany 1:250), anti-TRPM4 antibody (ab123936, Abcam, Cambridge, UK, 1:1000) and loading control alpha tubulin antibody (ab4074, Abcam, Cambridge, UK, 0.25 μ/mL) and subsequently for 1 h at room temperature with anti-rabbit IgG antibody (7074S, Cell Signaling Technology, Frankfurt, Germany, 1:10,000) and anti-mouse IgG antibody (7076S, Cell Signaling Technology, Frankfurt, Germany, 1:2500) diluted in 5% milk. The membranes were washed, and signals were detected by Immobilon Western Chemiluminescent HRP substrate (WBKLS0500, Millipore, Burlington, MA, USA) and scanned by INTAS Chemostar.