Pierce protein free pbs blocking buffer

Cells were lysed in 350 uL RIPA buffer and lysate was transferred to 1.5 ml tubes and centrifuged at 5000 x g for 5 minutes at 4°C. Total protein concentrations for each sample were determined using the Pierce BCA Protein Assay kit (ThermoFisher Scientific). Levels of AEBP1 and GAPDH were detected using western blot analysis. An equal amount of protein was loaded on a NuPage pre-cast Bis-Tris protein gel with a 4–12% polyacrylamide gradient (Life Technologies), and then transferred from gels to nitrocellulose membranes (Life Technologies). Membranes were placed in Pierce Protein-Free (PBS) Blocking Buffer (ThermoFisher Scientific) for ninety minutes at room temperature, and then incubated overnight at 4°C with primary antibodies directed against ACLP/AEBP1 (anti-mouse 1:1000 dilution; Santa Cruz; Dallas, TX; catalog number: sc-271374) or GAPDH (anti-rabbit 1:1000; Cell Signaling Technology; Danvers, MA). Membranes were washed for eight minutes in 1X TBS-Tween buffer, and then incubated with secondary antibodies, either anti-mouse or anti-rabbit, labeled with horseradish peroxidase (1:3000; Cell Signaling Technology) for one hour at room temperature. Membranes were incubated with Clarity Western ECL Blotting Substrate (Bio-Rad; Hercules, CA) and protein bands were imaged using the Odyssey FC imaging system (LI-COR Biotechnology; Lincoln, NE).

The equilibrium dissociation constant (K_d) values of ErbB2 aptamer were determined using an enzyme-linked oligonucleotide assay (ELONA). Briefly, 96-well ELISA plates (Corning^®, Sigma-Aldrich) were coated with ErbB2 (Cat. No. 1129-ER, R&D system) at 4 ºC for 16 h. The treated wells were blocked with Pierce^™ Protein-Free (PBS) Blocking Buffer (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h, followed by three washes with PBS. The biotin-labeled aptamers were added and incubated for 1 h at room temperature. After extensive washing with washing buffer (10 mM PBS, 0.05% Tween-20, pH 7.4), Pierce^™ High Sensitivity Streptavidin HRP (Thermo Fisher Scientific) was added to each well to bind biotin-conjugated aptamer. After incubation for 1 h at room temperature, the plates were washed again as described above. Tetramethyl benzidine (TMB) substrate solution (Thermo Fisher Scientific) was added to each well and incubated for 30 min at room temperature. Then, the reaction was quenched by addition of 2 M H₂SO₄. The protein-bound aptamer-streptavidin complexes were quantified by determining the absorbance at 450 nm using GloMax^® Discover System (Promega, Madison, WI, USA). The saturation curve was plotted and K_d was analyzed with Sigma Plot 12.5 (https://systatsoftware.com/products/sigmaplot/) software.

Serum samples in NSG mice with Nalm-6 13-3B5* were collected at day 5 and day 15 after target cell injection in K₂EDTA tubes for MuSK antibody ELISA. To detect human anti-MuSK IgG4, the histidine-tagged recombinant extracellular domain of human MuSK (aa 24–495, R&D Systems, catalog no. 10189-MK) was coated on ELISA plates in PBS overnight at 4 °C at a concentration of 5 µg ml⁻¹. Plates were washed with washing buffer (Invitrogen, catalog no. 00-0400-59), and blocked with Pierce Protein-Free (PBS) Blocking Buffer (Thermo Scientific, catalog no. 37572). Mouse serum samples were evaluated at a dilution of 1:50 to 1:100 in comparison to a 13-3B5 purified recombinant human monoclonal IgG4 antibody as a reference standard for quantitation. Antihuman IgG (H+L) HRP (Bethyl, catalog no. A80-119P) was used to detect human antibodies. Plates were protected from the light and placed in the dark for 2 h. After washing plates three times, 100 μl of TMB (Thermo Scientific, 34028) was added for 30 min. Plate reading were conducted using ELISA reader (Tecan, Infinite F-50) within 15 min after adding stop solution (Invitrogen, SS04).

Female 7–8 weeks old C57BL/6 mice (n = 4 per group) were intravenously administered with the equivalent dose (3.8 × 10¹⁰ vg/mouse) of either free AAV‐eGFP or RBC‐AAV‐eGFP. Four weeks after the initial AAV exposure, the same mice were intravenously injected with the equivalent dose (3.8 × 10¹⁰ vg/mouse) of either free AAV‐fLuc or RBC‐AAV‐fLuc. Blood was collected on days 0, 27, 42, and 56 post the first injection to quantify the amount of nAbs. nAb titer was measured by ELISA. In brief, 96‐well plate was coated with AAV‐eGFP (2.5 × 10⁹ vg/well in 100 µL PBS) overnight at 4 °C. Plates were then washed 6 times with washing buffer (PBS + 0.01% Tween‐20) and blocked using 300 µL of Pierce Protein‐Free (PBS) Blocking Buffer (Thermo Scientific) for 2 h. After washing the plates 6 times, 100 µL of serum at a serial dilution was added to each well and incubated for 2 h at room temperature. The plates were then washed again and goat anti‐mouse IgG HRP in 100 µL of PBS (1:10 000 dilution) was added to each well for incubation for 1 h. After washing the plates, 100 µL TMB substrate was added to each well; 10 min later, the reaction was stopped by adding 100 µL stopping buffer. Absorbance at 450 nm was measured using a plate reader. The antibody titer (EC₅₀) was defined as the dilution factor at which the absorbance value dropped to half of the maximum.

ACE2 (recombinant mouse (HEK293cells; cat#230-30172) and recombinant human ACE2 (HEC 293 cells; cat# 230-30165), RayBiotechLife, Inc., Peachtree Corners, GA), dissolved in carbonate/bicarbonate buffer, was immobilized (25 μg ml⁻¹) on glass slides for 1 h at room temperature (RT), blocked with a non-protein buffer (Pierce Protein-free (PBS) blocking buffer, Cat# 37572, ThermoFisher Scientific), washed, incubated with SP-555 at different concentrations (0.01–1 μg ml⁻¹) in Hank’s buffered salt solution (HBSS) for 1 h at RT, washed, mounted, and imaged. SP-555 intensity from ten fields in two slides for each concentration were analyzed and expressed as intensity per μm².