Hrp conjugated anti rabbit igg secondary antibody

A codon-harmonized form of the full length PfHsp70-z (PlasmoDB accession no. PF3D7_088000) which we previously described was used for the expression of recombinant PfHsp70-z protein using Escherichia coli JM109 cells following a previously described method (Zininga et al. 2015a (link)). The purified protein was extensively dialyzed overnight at 4 °C against a storage buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.8 mM DTT, 10 % (v/v) glycerol). Protein concentrations were estimated by Bradford assay. The presence of PfHsp70-z was confirmed using both rabbit anti-His, rabbit polyclonal peptide anti-PfHsp70-z antibodies, and HRP-conjugated anti-rabbit IgG secondary antibodies (1:2000) (ThermoScientific, USA). Imaging of the protein bands on the blot was conducted using the ECL kit as per manufacturer’s instructions. Images were captured using ChemiDoc Imaging system (Bio-Rad, USA).

SF2 cells transfected with either knockdown miR-1 or scramble probes were lysed in M-per buffer plus protease inhibitor cocktail (Sigma-Aldrich). The samples were centrifuged (14000 rpm, 5 min, 4°C), and the supernatants were diluted in NuPage LDS buffer and then separated on a 4–20% NuPage Bis-tris gradient gel (Thermo Fisher Scientific). Separated proteins were electro-transferred on to PVDF membranes (Thermo Fisher Scientific). After blocking with 3% non-fat skim milk in PBS, the membrane was probed with anti-Cx43 (Abcam, United Kingdom) or anti-β-actin primary antibodies (1:100 dilution, Abcam) in PBS. The primary antibodies were detected using HRP-conjugated anti-rabbit IgG secondary antibodies (1:500 dilution, Thermo Fisher Scientific) using an ECL kit (Amersham Biosciences Co., NJ, United States) and a LAS 4000 UV mini system (Fujifilm-GE healthcare, Japan).

rAAV-RFP-infected retinas or control retinas were digested with cocktail containing 1 mg ml⁻¹ collagenase D (Roche) and 30 μg ml⁻¹ DNase I (Sigma-Aldrich) in RPMI at 37 °C for 45 min. Tissues were pipetted to break tissue down and filtered through a 70-μm filter. Then, cells were lysed in RIPA buffer and boiled for 5 min with sample buffer. Western blotting was carried out in a similar manner to that previously reported^{9 (link)}. In brief, 15% gels were used and run at 10 mA per gel for 30 min and 40 mA per gel until ladder separation. Wet transfer was carried out at 120 mA per gel for 90 min on ice. RFP-Tag rabbit polyclonal antibodies (OriGene Technologies, catalogue no. AP09229PU-N) were used at a concentration of 1:1,000 and incubated overnight in a cold room. After being washed, HRP-conjugated anti-rabbit IgG secondary antibodies (Thermo Fisher, G-21234) were used at a concentration of 1:5,000 at room temperature for 2 h and imaged using the ChemiDoc MP imaging system (Bio-Rad).