Anti rabbit igg hrp conjugate

The D5 peptide (10 µg and 20 µg) and H4 peptide (10 µg and 20 µg) were attached to a nitrocellulose membrane by applying it drop by drop. The membrane was blocked with 5% nonfat dry-milk solution in TBT-T for 1h and then washed with TBT-T. Membranes were incubated with 1 mg/mL Hc-TeNT for 1h, and afterward, primary antibodies diluted in a BSA 5% solution were added o/n at 4 °C. Antibodies used were mouse anti-polyhystidine (1:1000; 27-471001; Amersham Pharmacia Biotech, Buckinghamshire, UK), rabbit anti-TrkB (1:1000; ab33655; Abcam, Cambridge, UK) and mouse anti-β-tubulin (1:10,000; 556321; BD Biosciences). Secondary antibodies used were anti-rabbit IgG HRP conjugate (Thermo Scientific), and anti-mouse IgG HRP conjugate (Bio-Rad Laboratories, Hercules, CA, USA) diluted 1:3000 in TBS-T plus 5% nonfat dry-milk. Membranes were developed using a 1:1 chemoluminiscent mixture [1 M Tris-HCl pH 8.5, 0.5 M luminol, 79.2 mM p-coumaricacid: 1 M Tris-HCl pH 8.5, 8.8 M H₂O₂], and exposed to ECL film membranes (Amersham Pharmacia Biotech Buckinghamshire, UK). Analysis of the resulting dots was performed using Quantity One (Bio-Rad Laboratories, Hercules, CA, USA).

For GUS staining, protein extracts were prepared from 10 mg of seedlings in 10 µl of SDS sample buffer. All of the eluate was loaded onto a 4–12% SDS-PAGE gel (Invitrogen) and the gel was run in a Bolt Mini Gel tank (Thermo Fisher Scientific). The resulting gel was subjected to immunoblot analysis using the iBlot2 dry blotting system (Thermo Fisher Scientific). Rabbit polyclonal anti-HSP22 (Eurofins Genomics) (1:1000 diluted), anti-HSP17.6 (ab80183; Abcam) (1:1000 diluted), and anti-HSP21 (ab80175; Abcam) (1:1000 diluted) and anti-rabbit IgG HRP conjugate (1:5000 diluted, Thermo Fisher Scientific) were used as primary and secondary antibodies, respectively. Signals were detected using chemiluminescence HRP substrates (Millipore) and an image analyser (LAS4000, GE healthcare). Protein size was determined by MagicMark XP (Thermo Fisher Scientific). Coomassie Brilliant Blue (CBB)-stained membranes were used as loading controls. The signal intensity of each band was quantified by ImageJ (NIH). Values in graphs are mean ± SEM. Three independent experiments were performed. Statistical significance was computed using a one-way ANOVA test followed by a post-hoc Tukey’s HSD test (https://astatsa.com/OneWay_Anova_with_TukeyHSD/).

For immunoblot analysis, protein extracts were prepared from petals at the indicated position in 100 µL of SDS sample buffer. All of the eluates were loaded onto 4–12% SDS–PAGE gel (Invitrogen) and separated in a Blot Mini Gel tank (Thermo Fisher Scientific). The resulting gel was subjected to immunoblotting using an iBlot2 dry-blotting system (Thermo Fisher Scientific). Rabbit polyclonal anti-GFP (ab290, Abcam, 1:1,000 diluted), anti-histone H3 (ab1791, Abcam, 1:1,000 diluted), and anti-rabbit IgG HRP conjugate (1:5,000 diluted, Thermo Fisher Scientific) were used as primary and secondary antibodies. Signals were detected with chemiluminescence HRP substrates (Millipore) and Image analyzer (LAS4000, GE healthcare). Molecular mass was determined by MagicMark XP (Thermo Fisher Scientific). After detection of signals by LAS4000, each membrane was dried, washed with PBS, and stained with Coomassie brilliant blue at room temperature for 30 min (CBB: Thermo Fisher Scientific). The resulting membrane was scanned using an ApeosPort C4570 (FUJIFILM). Histone H3 or CBB staining was used as a loading control. Signal intensity of each band was quantified in ImageJ (NIH). Statistical significance was computed using Student’s t test in Microsoft Excel (Microsoft 365 16.61).