Hrp conjugated rabbit antigoat igg h l

Primary antibodies used in this study were polyclonal rabbit anti-DDDDK tag (MBL, PM020), polyclonal goat anti-Dynamin2 (C-18) (sc-6400, Santa Cruz Biotechnology), monoclonal mouse anti β-actin (A5441, Merck), and monoclonal rabbit anti-GFP (D5.1) XP (2956, CST). All the secondly antibodies used in this study, Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H + L) (A21206), Alexa Fluor 555-conjugated donkey anti-rabbit IgG (H + L) (A31572), Alexa Fluor 568-conjugated donkey antigoat IgG (H + L) (A11057), HRP-conjugated rabbit antigoat IgG (H + L) (31402), HRP-conjugated rabbit antimouse IgG (H + L) (31450), HRP-conjugated goat anti-rabbit IgG (H + L) (31460) were purchased from Thermo Fisher Scientific.

The rHc8-specific antibody levels were ascertained by ELISA. The appropriate rHc8 concentration (250 ng/μl) and the optimal dilution of goat serum (1: 200) were determined by chessboard titration. In brief, 100 μl of rHc8 diluted in carbonate coating buffer (0.05 M, pH 9.6) was coated on 96-well microliter plates at 4°C overnight. Skim milk (5%) in PBS containing 0.5% Tween 20 (PBST) was used to block the plates at 37°C for 1 h. As to the primary antibody, diluted goat serum in PBST was incubated at 37°C for 1 h, followed by washing three times. The horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG (H + L) (Thermo Scientific, Waltham, MA, USA) was then used in l:5,000 dilution at 37°C for 1 h. After five washes, the colorimetric reaction was initiated with 3, 3′, 5, 5′-tetramethylbenzidine (TMB) substrate. The substrate reactions were terminated with 2 M sulfuric acid, and absorbance values at 450 nm (OD450) were measured by a microplate reader (Thermo Scientific).