Matrigel-matrix-coated plates (BD Biosciences, cat. # 354607) were incubated with 100% SBlock (Pierce, cat. # 37538), for 10 min at room temperature. One hundred microlitres of varying amounts recombinant proteins diluted in 50% of SBlock in PBS was added and incubated at RT for 2 h. After washing four times with 400 μl of wash buffer (10% SBlock, 0.1% Tween-20 in PBS), 100 μl of HRP-conjugated goat anti-human IgG-Fc (1:10,000 dilution in 50% of SBlock in PBS; Jackson Immunoresearch, cat. # 109-035-098) was added in each well and the plate was incubated at RT for 2 h. The plate was washed four more times with 400 μl of wash buffer, before addition of 50 μl of TMB solution (Thermo Scientific, cat. # 34028) and incubation at RT for 30 min. The reaction was stopped with stopping solution (Sigma, cat. # S5814), and the absorbance at 450 nm was measured using an ELISA reader (Bio-Rad).
Sblock
Manufactured by Jackson ImmunoResearch
SBlock is a laboratory product designed for use in biotechnology and scientific research applications. It functions as a blocking agent, intended to reduce non-specific binding in immunoassays and other related experiments. The core purpose of SBlock is to minimize background signals and improve the accuracy and reliability of experimental results.
Automatically generated - may contain errors
2 protocols using sblock
1
Quantitative Protein-Binding Assay
2
VEGF-trap Enzyme-linked Immunosorbent Assay
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Enzyme-linked immunosorbent assay for VEGF-trap was developed as has been previously described (Koh et al, 2010 (link)). A 96-well plate was coated overnight at 4°C with 200 ng of VEGF in 100 μl of PBS. The plate was washed four times with 400 μl wash buffer [PBS, 10% SBlock (Pierce, cat. # 37538), 0.05% Tween-20] and then coated for 2 min with SBlock at RT. Fifty microlitres of trap standards, samples and controls were added along with 50 μl of incubation buffer (100% SBlock, 0.1% Tween-20) and incubated on the bench, at RT for 2 h. After washing four times with 400 μl of wash buffer, 50 μl of HRP-conjugated goat anti-human IgG-Fc (1:10,000 dilution in PBS; Jackson Immunoresearch, cat. # 109-035-098) along with 50 μl of incubation buffer was added in each well and the plate was incubated at RT for 2 h. The plate was washed four more times with 400 μl of wash buffer, before addition of 50 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Thermo Scientific, cat. # 34028) and incubation at RT for 30 min. The reaction was stopped with stopping solution (Sigma, cat. # S5814), and the absorbance at 450 nm was measured using an ELISA reader (Bio-Rad).
Human VEGF was measured using a commercially available sandwich ELISA (R&D systems, cat. # DVE00) following the manufacturer's protocol. One hundred microlitres of tissue supernatant was used for each sample.
Human VEGF was measured using a commercially available sandwich ELISA (R&D systems, cat. # DVE00) following the manufacturer's protocol. One hundred microlitres of tissue supernatant was used for each sample.
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