To begin, culture and prepare the HEK 293 T-cells expressing RapR-Shp2 for immunoprecipitation. For preparing the Sepharose, add resuspended Protein G-Sepharose into a 1.5 milliliter tube. After adding the lysis buffer, micro-centrifuge the tube for one minute at 2000 G and carefully remove the lysis buffer.
Then resuspend the Sepharose in 380 microliters of lysis buffer by inverting the tube a few times. Incubate the Sepharose with BSA and the antibody on a rotator at four degrees Celsius for 1.5 hours. After washing the Sepharose as demonstrated earlier, use a cut tip to carefully resuspend it in the lysis buffer.
Add the supernatant from the prepared HEK 293 T-cells expressing RapR-Shp2 to 50 microliters of the suspended Sepharose in a new 1.5 milliliter tube, and incubate the tube on a rotator at four degrees Celsius for 1.5 to two hours.
