Levels of TSG-6 protein in the medium collected from hASC were determined by ELISA as previously described with modifications [22 (link)]. A 96-well plate (Maxisorp; Nunc) was coated with 50 μl of 10 μg/ml TSG-6 antibody (clone A38.1.20; Santa Cruz Biotechnology) in 0.2 M sodium bicarbonate buffer (pH 9.2) overnight at 4°C. Plates were washed with washing buffer (R&D systems) after this and all subsequent steps. Nonspecific sites were blocked with 0.25% BSA in PBS/0.05% Tween (blocking buffer) for 1 hr at room temperature. Samples of 50 μl or standards of rhTSG-6 protein (R&D systems) in dilution buffer were added and incubated for 2 hrs at room temperature, followed by 50 μl/well of 0.5 μg/ml biotinylated anti-hTSG-6 antibody (R&D systems) in blocking buffer for 2 hrs at room temperature. Bound antibody was detected by incubation for 20 min with streptavidin-horseradish peroxidase (R&D systems), diluted 1:200 in PBS and then with substrate solution (R&D systems) for 20 min. Absorbance at 450 nm and 584 nm was measured by spectrophotometer (SpectraMax M5; Molecular Devices, Sunnyvale, CA, http://www.moleculardevices.com/ ).
Tsg 6 antibody clone a38
Manufactured by Santa Cruz Biotechnology
The TSG-6 antibody (clone A38.1.20) is a laboratory tool used to detect and quantify the presence of the TSG-6 protein in biological samples. TSG-6 is a secreted protein involved in various cellular processes. This antibody can be utilized in techniques such as Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) to study the expression and localization of TSG-6 in research settings.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin hrp, by R&D Systems (2 mentions)
Biotinylated anti human tsg 6 antibody, by R&D Systems (2 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Streptavidin horseradish peroxidase, by R&D Systems (1 mentions)
Maxisorp, by Thermo Fisher Scientific (1 mentions)
Washing buffer, by R&D Systems (1 mentions)
Substrate solutions, by R&D Systems (1 mentions)
Tmb substrate, by Cell Signaling Technology (1 mentions)
3 protocols using tsg 6 antibody clone a38
1
Quantification of TSG-6 Protein in hASC Medium
2
TSG-6 Protein Quantification in MSCs
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TSG-6 protein level in the supernatant of human MSCs cultured with inflammatory cytokines was measured by ELISA as previously described [17 (link)]. Briefly, a 96-well plate was coated with 50 μl of 10 μg/ml TSG-6 antibody (clone A38.1.20, Santa Cruz Biotechnology) overnight, in a coating buffer of 0.2 M sodium bicarbonate (pH 9.2). The plate was washed with PBS, blocked with 0.25% BSA and 0.05% Tween 20 in PBS for 30 min, and again washed with PBS. Samples (50 μl) and a standard of human recombinant TSG-6 protein (R&D Systems) in blocking buffer were added and incubated at 4 °C overnight. Then, the plate was washed with PBS, followed by addition of biotinylated anti-human TSG-6 antibody (50 μl of 0.5 μg/ml; R&D Systems) and incubated for 3 h at room temperature. After washing with PBS, streptavidin–HRP (50 μl; R&D Systems) was added for 30 min at room temperature, and developed using substrate solutions (R&D Systems).
3
Quantification of TSG-6 Protein in MSCs
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The TSG-6 protein level in the medium of human MSCs cultured with inflammatory cytokines was measured by ELISA as previously described [17 (link)]. Briefly, a 96-well high binding plate was coated with 50 μl of 10 μg/ml TSG-6 antibody (clone A38.1.20, Santa Cruz Biotechnology) overnight, in a coating buffer of 0.2 M sodium bicarbonate (pH 9.2). The plate was washed with PBS, blocked with blocking buffer (0.25% BSA and 0.05% Tween 20 in PBS) for 30 min. Samples (100 μl) and a standard of human recombinant TSG-6 protein (R&D Systems) in blocking buffer were added and incubated at room temperature for 2 h. Then, the plate was washed with PBST (0.05% Tween 20) three times, followed by the addition of a biotinylated anti-human TSG-6 antibody (50 μl of 0.5 μg/ml; R&D Systems) and incubated for 2 h at room temperature. After washing with PBST three times, streptavidin–HRP (50 μl; R&D Systems) was added for 30 min at room temperature and developed using TMB substrate (Cell Signaling Technology).
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