| Assay Method Information | |
| | Alpha Screen Assay |
| Description: | Measurement of various protein-protein interactions was performed using the Alpha Screen technology developed by Perkin Elmer. Recombinant RAS proteins (H-, N-, K-RAS variants) carried a c-terminal Avi-tag used for biotinylation. Interacting proteins (SOS1, PI3K and CRAF) were expressed as glutathione S transferase (GST) fusions. Accordingly, the Alpha Screen beads were glutathione coated Alpha Lisa acceptor beads (Perkin Elmer AL 109 R) and Alpha Screen Streptavidin conjugated donor beads (Perkin Elmer 6760002L). Nucleotides were purchased from Sigma (GTP #G8877, GDP #G7127), Tween-20 from Bio-Rad (#161-0781). All interaction assays were carried out in PBS, containing 0.1% bovine serum albumin, 0.05% Tween-20 and 10 μM of the corresponding nucleotide. Assays were carried out in white ProxiPlate-384 Plus plates (Perkin Elmer #6008280) in a final volume of 20 μl. In brief, biotinylated RAS proteins (10 nM final concentration) and GST-SOS1, GST-PI3K or GST-CRAF (10 nM final) were mixed with glutathione acceptor beads (5 μg/ml final concentration) in buffer, containing the corresponding nucleotides (GDP or GTP for assays containing SOS1, only GTP for interaction assays containing PI3K or CRAF) and were incubated for 30 min at room temperature. After addition of strepavidin donor beads (5 μg/ml final concentration) under green light, the mixture was further incubated for 60 min in the dark at room temperature. |
| Affinity data for this assay | |
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