Bait and prey proteins were normalised to activities suitable for the AVEXIS assay as described49 . Biotinylated baits that had been either purified or dialysed against HBS were immobilised in streptavidin-coated 96-well microtitre plates (NUNC). Preys were incubated for two hours, washed 3x HBS/0.1 % Tween-20, 1x HBS and 125 μg/mL of nitrocefin added, and absorbance values measured at 485 nm on a Pherastar plus (BMG laboratories). A protein consisting of the Cd4d3+4 tag alone was used as a negative control bait and a biotinylated anti-Cd4 monoclonal antibody (anti-prey) used as a positive control as required. The mouse Izumo1-Juno interaction has been repeated more than three times in the laboratory by AVEXIS using independent protein preparations; importantly, the interaction is observed if the Izumo1 or Juno proteins are presented as either the bait or prey. Similarly, the Izumo1-Juno interactions from different mammalian species were repeatable and independent of the bait-prey orientation.
Streptavidin coated 96 well microtitre plates
Manufactured by Thermo Fisher Scientific
Streptavidin-coated 96-well microtitre plates are designed for use in various bioassays and immunoassays. The plates are coated with the protein streptavidin, which has a high affinity for the molecule biotin. This allows for the capture and immobilization of biotinylated molecules, such as proteins, DNA, or other biomolecules, onto the plate surface.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using streptavidin coated 96 well microtitre plates
1
AVEXIS Assay for Protein-Protein Interactions
2
ELISA-based Protein-Protein Interaction Screening
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bait and prey proteins were normalized to activities that have been previously shown to detect transient interactions [12 (link)] and screened using the ELISA-based AVEXIS methodology as described in [14 (link)]. Briefly, biotinylated bait proteins were immobilized on streptavidin-coated 96-well microtitre plates (Nunc) and washed with HBS. Normalized β-lactamase-tagged preys were incubated for 1 h, the wells were washed with HBS and finally 125 µg ml−1 of the β-lactamase substrate, nitrocefin, was added. Absorbance values were measured at 485 nm on a Pherastar Plus (BMG Laboratories). A bait protein consisting of the CD4d3+4 tag alone was used as the negative control. All steps were done at room temperature. The assays were repeated three times using independent protein preparations.
3
AVEXIS Assay for Protein-Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bait and prey proteins were normalised to activities suitable for the AVEXIS assay as described49 . Biotinylated baits that had been either purified or dialysed against HBS were immobilised in streptavidin-coated 96-well microtitre plates (NUNC). Preys were incubated for two hours, washed 3x HBS/0.1 % Tween-20, 1x HBS and 125 μg/mL of nitrocefin added, and absorbance values measured at 485 nm on a Pherastar plus (BMG laboratories). A protein consisting of the Cd4d3+4 tag alone was used as a negative control bait and a biotinylated anti-Cd4 monoclonal antibody (anti-prey) used as a positive control as required. The mouse Izumo1-Juno interaction has been repeated more than three times in the laboratory by AVEXIS using independent protein preparations; importantly, the interaction is observed if the Izumo1 or Juno proteins are presented as either the bait or prey. Similarly, the Izumo1-Juno interactions from different mammalian species were repeatable and independent of the bait-prey orientation.
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!