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/ ).
Washing buffer
Manufactured by R&D Systems
Washing buffer is a lab solution used to wash and rinse samples during various experimental procedures. It helps remove unwanted materials from the sample, preparing it for further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax m5, by Molecular Devices (1 mentions)
Streptavidin horseradish peroxidase, by R&D Systems (1 mentions)
Maxisorp, by Thermo Fisher Scientific (1 mentions)
Tsg 6 antibody clone a38, by Santa Cruz Biotechnology (1 mentions)
Bio plex 200 system, by Bio-Rad (1 mentions)
Human magnetic luminex assay lxsahm, by R&D Systems (1 mentions)
2 protocols using washing buffer
1
Quantification of TSG-6 Protein in hASC Medium
2
Quantifying BDNF and CNTN1 in Plasma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood samples were collected at 8:00 a.m. from participants who had fasted overnight. The BDNF and CNTN1 levels in plasma samples were obtained using the Human Magnetic Luminex Assay (LXSAHM; R&D Systems, Minneapolis, MN, USA). The assays were conducted according to the protocol that was provided by R&D Systems. Briefly, a 96-well filter-bottom microplate (Millipore, Billerica, MA, USA) was blocked for 10 min with assay buffer (R&D Systems). To generate standard curves, four-fold serial dilutions of standards were prepared in serum diluent (R&D Systems). 50 μL of standards and plasma samples (1:2 dilution) were added to the wells, which contained 50 μL of the immunobead mixture. The microplate was incubated for 40 min at room temperature in the dark and then washed three times with washing buffer (R&D Systems) using a vacuum manifold. A mixture of biotin-conjugated secondary antibodies (R&D Systems) was added. After 40 min of incubation and three washes, streptavidin-phycoerythrin (R&D Systems) was added. After 10 min, the immunobeads were washed three times, resuspended in 50 μL assay buffer, and analyzed using the Bio-Plex 200 system (Bio-Rad Laboratories, Hercules, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!