3 add 4 ml ap fusion protein see note 12 and incubate

This preview shows page 336 - 338 out of 372 pages.

3.Add 4 mL AP fusion protein (seeNote 12) and incubate at room temperature for 90min (seeNote 22). Swirl briefly to mix at approximately the 30 min and 60 mintime-points.4.Remove the AP fusion protein solution with a pipet (seeNote 13). Wash cells 6×with 10 mL cold HBAH (seeNote 21). For each wash, incubate HBAH with cellsfor 5 min and gently swirl the medium by hand or on a platform shaker.5.Aspirate the HBAH and add 10 mL acetone-formalin fixative slowly and swirl for15 s exactly. Longer fixation might destroy some AP activity.6.Aspirate the fixative and wash 2×with 10 mL HBS. Leave 10 mL HBS on the plate.7.Incubate the plate containing 10 mL HBS on a flat shelf in a 65°C preheated ovenfor 100 min (seeNote 23).8.Wash with 10 mL AP staining buffer.9.Add 4 mL BCIP/NBT substrate. Incubate at room temperature under a shade ofaluminum foil. Staining can be monitored periodically against a white backgroundunder a dissecting microscope (seeNote 19). Color should become visible in about30 min. Sometimes it takes a few hours, or can even be incubated overnight, butbackground color will begin to appear.10.Stop the reaction when it looks good by washing the plate with PBS and store thecells in 10 mL PBS with 10 mMEDTA at 4°C in the dark.3.8. Quantitative Assayfor Receptor-AP Fusion Binding to Cell SurfacesThis is the method of choice to screen cell lines for potential expression of aligand that is cell surface associated (or extracellular matrix associated). It can
Cloning and Characterization of RTK Ligands325also be used to study quantitative aspects of ligand–receptor interactions, suchas equilibrium constants or rate constants of binding, or the effects of antagonists,cofactors, or mutations.1.Grow cells to be tested until they are almost confluent, or have just reached confluence.This can be done in a six-well tissue-culture plate.2.Wash cells once with 3 mL cold HBAH (seeNote 21).3.Add 1 mL AP fusion protein solution (seeNote 12) and incubate at room temperature for90 min (seeNote 22). Swirl briefly to mix at approx the 30 min and 60 min time-points.4.Remove the AP fusion protein solution with a pipet (seeNote 13). Wash cells 6×with 5mL cold HBAH (seeNote 21). For each wash, incubate HBAH with cells for 5 min andgently swirl the medium by hand or on a platform shaker.5.Aspirate out all the remaining HBAH completely, and lyse cells with 300μL Triton-Tris buffer at room temperature. It usually takes a few minutes at most for the cells todissolve.6.Collect all the lysate to an Eppendorf tube, rinse the plate with an additional 200μLTriton-Tris, and pool this with the first lysate. Vortex for 30 s, allow to sit at roomtemperature for 5 min, and vortex again.7.Spin down the lysate at maximum speed in a microcentrifuge, and transfer thesupernatant to another Eppendorf tube.

Upload your study docs or become a

Course Hero member to access this document

Upload your study docs or become a

Course Hero member to access this document

End of preview. Want to read all 372 pages?

Upload your study docs or become a

Course Hero member to access this document

Term
Fall
Professor
DINNER
Tags
Alastair D Reith

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture