Briefly, cells were lysed for 5 minutes with supplied lysis buffer in an orbital shaker at 700 rpm, then ATP detection substrate solution was added to the wells and agitated for 5 minutes.
The nuclei were then spun down briefly and resuspended in buffer B (20 mM Hepes pH 7.9, 40 mM NaCl, 1.5 mM MgCl2, 20 v/v glycerol, 0.2 mM EDTA), and agitated for 30' at 4°C.
The suspensions were combined and agitated for 30 s and then immediately loaded into the cytometer.
Five millilitres of the filtrate was introduced into a test tube and agitated for 10 s.
The supernatant was immediately blended with Ni-NTA resin (Novagen), pre-equilibrated with the binding buffer, and agitated at 4 °C for 1 h.
Subsequently, membranes were blocked with 5% (w/v) milk powder in 0.05% Tween-20 buffer and slowly agitated for 1 hr at room temperature.
Peptides were incubated with equimolar CLR01, CLR03, or buffer and agitated at 1400 rpm at 37°C.
Sensor tips were pre-wet for 15 mins in buffer immediately prior to use, and the microplates were filled with 200 μl per well of diluted samples (VEGF) or buffer and agitated at 1000 rpm.
Lyophilized PAP248-286(Ala) was dissolved in PBS (100 µM), incubated with CLR01 (100 µM), CLR03 (100 µM), or buffer and agitated at 1400 rpm at 37°C.
All microarrays were stripped by pouring boiling 0.5% SDS over them and agitating for one hour.
