Horseradish peroxidase conjugated goat anti mouse igg antibody

Immunoblotting was performed as described by Ahn et al. (2011) (link) and Cho et al. (2013) (link), using mouse monoclonal antibodies against the Flag tag (Sigma), GFP (Clontech), RPL10a (Santa Cruz Biotechnology), and histone H3 (Santa Cruz Biotechnology). The immunoblots were treated with horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (Invitrogen), followed by signal detection using Imagequant LAS 4000 (GE Healthcare Life Sciences).

SYK-6 cells harbouring pJB-tonB2His that carried tonB2 fused with a His6 tag at its C-terminus in pJB861 were grown in LB containing Km and 1.0 mM m-toluate to an OD₆₀₀ of 0.5. The outer membrane fraction was prepared according to the method reported by Hashimoto et al.^{52 (link)}. The cells were harvested by centrifugation at 4800 × g for 10 min, washed twice with water and incubated with 26 mM Tris-HCl buffer (pH 8.3) containing 438 mM sucrose, 1.5 mM EDTA and 0.22 mg ml⁻¹ lysozyme at 30 °C for 1 h. The resulting solution was centrifuged at 19,000 × g for 60 min to remove spheroplasts, and then the supernatant was ultracentrifuged at 120,000 × g for 60 min to obtain the outer membrane fraction. The total membrane fraction was prepared as described above. Western blot analysis was performed against the prepared outer membrane fraction and total membrane fraction using anti-DdvT and anti-His6 antibodies (Invitrogen, 1.0 µg ml⁻¹) as primary antibodies. Horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (Invitrogen, 0.04 µg ml⁻¹) were used as the secondary antibodies for the anti-His6 antibodies.

Protein extracts (30 µg) were subjected to SDS-PAGE and immunoblotting as described previously (Ahn et al., 2011 ; Lee et al., 2013 (link)). Anti-Tap46 antibodies were generated in rabbits against an oligopeptide, NQPLIFGPASIVGGP, that corresponds to amino acid residues of Arabidopsis Tap46 at positions 289 to 303 using the antibody production services of Cosmogenetech (http://www.cosmogenetech.com). Immunoblotting was performed using mouse monoclonal antibodies against the haemagglutinin (HA) tag (1:10 000 dilution; Applied Biological Materials), the Myc tag (1:10 000 dilution; Applied Biological Materials), and the Flag tag (1:10 000; Sigma), or using rabbit polyclonal antibodies against α-tubulin (1:1000 dilution; Sigma), the PP2A catalytic subunit (1:1000; Cell Signalling), and Tap46 (1:5000; Cosmogenetech). The membranes were then treated with horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (1:10 000; Invitrogen) or goat anti-rabbit antibodies (1:10,000; Invitrogen), respectively. Signals were detected on X-ray film (Kodak) using an ECL chemiluminescence kit (ELPIS-Biotech, Inc.).

The extraction and quantification of total protein were carried out from 1 g of frozen cassava roots according to Maass et al. [88 (link)] with modifications of Cuellar et al. [89 (link)]. Proteins were resolved on a 12% SDS-polyacrylamide gel and blotted to 0.45 μM PVDF membranes (ThermoFischer). The blotting was conducted in a Mini Trans-Blot^® Cell (Bio-Rad) apparatus. Membranes were blocked with 5% skim milk powder in Tris-buffered saline, washed, and incubated in Tris-buffered saline plus 0.1% Tween 20 with either the polyclonal antibodies anti-PSY [56 (link)] or anti-OR [51 (link)]. After washing, the membranes were incubated for 1 h with a horseradish peroxidase- conjugated goat anti-Rabbit IgG antibody (Invitrogen, Cat.# G-21234) in a 1% skim milk solution in Tris-buffered saline plus 0.1% Tween 20. The membranes were washed and immunoblots were developed with Pierce^™ ECL Western Blotting Substrate (ThermoFischer). After striping the membrane with peroxidase [90 (link)], an anti-actin antibody (Sigma-Aldrich, Cat.#A0480) and a horseradish peroxidase-conjugated goat anti-Mouse IgG antibody (Invitrogen, Cat.# G-21040) were used to reprobe the immunoblot. Protein signals were quantified with ImageJ [91 (link)].

EIA for anti-S and anti-RBD mAbs was performed as we described previously with modifications (To et al., 2021 (link)). Briefly, 96-well Immuno plates (Nunc, Roskilde, Denmark; Cat# 243656) were coated with 100 μL/well (0.1 μg/well) of His-tagged SARS-CoV-2 S or RBD in 0.05 M carbonate bicarbonate buffer (pH 9.6) overnight at 4°C and then followed by incubation with a blocking buffer. After blocking, mAbs were diluted in a series of 2-fold dilution from 1 μg/ml. Then 100 μl diluted mAbs was added to the wells and incubated at 37°C for 1 hour. The attached mouse IgG was detected using horseradish-peroxidase-conjugated goat anti-mouse IgG antibody (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA; Cat# 31430). The reaction was developed by adding diluted 3,3’,5,5’-tetramethylbenzidine single solution and stopped with 0.3 N H₂SO₄. The optical density (OD) was read at 450 and 620 nm.

Treated CHSE/F cells were lysed in RIPA buffer containing protease inhibitor cocktail (Sigma-Aldrich, San Luis, Missouri, MO, USA). Proteins were denatured at 95 °C for 5 min, separated by SDS-polyacrylamide gel electrophoresis (10–12%), and then were transferred to nitrocellulose membranes (Millipore, Burlington, MA, USA). The membrane was incubated with PBS 1× containing 5% dehydrated skim milk at 4 °C. The membrane was incubated with anti-mCherry (1:2000, Abexxa, Cambridge, UK), anti-Flag antibody (1:2000, Millipore), anti-H2B antibody (1:2000, Abcam, Cambridge, UK), followed by horseradish peroxidase conjugated goat anti-mouse IgG antibody (1:5000, Invitrogen, Carlsbad, CA, USA). Bands on X-O-mat Blue films (AGFA) were visualized via enhanced chemiluminescence (ECL detection kit; Amersham, Little Chalfont, UK) according to the manufacturer’s instructions.