Pierce protein free tbs blocking buffer

Wells of a 96-well Nunc Maxisorp plate were coated with 200 μL
of 100 μg/mL dimethyl casein (C9801, Sigma-Aldrich) in HBS for
16 h at 4 °C.^{35 (link)} Wells were then blocked
with 300 μL Pierce protein-free TBS blocking buffer (37570,
Thermo Scientific) for 1 h at 25 °C. Wells were washed thrice
with 300 μL of HBS + 0.5% (v/v) TWEEN 20. Wells were then incubated
with 200 μL of TG2 variant at the indicated concentration in
HBS, 1 mM DTT, 1 mM EZ-Link Pentylamine-Biotin (21345, Thermo Scientific),
and 1 mM CaCl₂ for 30 min at 37 °C. Wells were washed
thrice with 300 μL of HBS + 0.5% (v/v) TWEEN 20 before incubation
with 150 μL of 0.3 μg/mL Pierce high-sensitivity streptavidin-horseradish
peroxidase (HRP) (21130, Thermo Scientific) diluted in HBS for 1 h
at 25 °C. Wells were washed six times in HBS + 0.5% (v/v) TWEEN
20, before signal generation by adding 100 μL of 1-Step Ultra
TMB-ELISA Substrate Solution (34029, Thermo Scientific). The reaction
was stopped by addition of 100 μL of 1 M HCl, and A₄₅₀ was measured on a FLUOstar Omega plate reader (BMG
Labtech).

Protein samples were mixed with Laemmli Sample Buffer (Bio-Rad, Hercules, USA) containing 10% (v/v) β-mercaptoethanol and heated to 95 °C for 10 min. Samples were subjected to electrophoresis on 10%, 12%, or 7.5% SDS-PAGE. Proteins were stained using Coomassie Blue Staining. For western blotting, proteins are separated by SDS-PAGE, transferred to PVDF transfer membrane, 0.45 µm (Thermo Fisher Scientific, Massachusetts, United States). Membranes were blocked with Pierce Protein-Free (TBS) Blocking Buffer (Thermo Fisher Scientific, Massachusetts, United States) for one hour, then incubated with the following primary antibodies: pc-asp anti-asprosin antibody, 1:5000 (0.2 µg/ml), mab anti-asprosin antibody (clone Birdy-1), 1:1000 (1 µg/ml), fibrillin-1 (rF90) 1:5000 (0.5 µg/ml), fibrillin-1 rabbit monoclonal antibody (CPTC-FBN1-3) 1:2500 dilution (1 µg/ml). and secondary antibody, mouse anti-rabbit IgG-HRP conjugate or goat anti-mouse IgG-HRP conjugate, 1:5000. All antibodies were diluted in blocking buffer. Signals were developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Massachusetts, USA). Blotted PVDF membranes used for Fig. 1F were cut and incubated with indicated primary antibodies.