As the func,onality of the probe was now validated against purified protein, we next wanted to inves,gate whether the PI5P photoaffinity probe 9 would be func,onal in cell lysate. Following addi,on of the probe to Expi293 cell lysate, and subsequent crosslinking and click reac,on with the TAMRA-azide fluorophore, it was observed that the PI5P diazirine probe 9 was capable of binding to mul,ple proteins within the complex cell lysate (Figure ). We could, however, not establish binding to endogenous hUHRF1, poten,ally due to low levels of endogenous expression compared to other factors. Thus, the same experiment was repeated using Expi293 cells overexpressing hUHRF1. However, even overexpressed levels of hUHRF1 were too low to allow effec,ve visualiza,on with the diazirine probe (Figure ). Ar,ficially increasing the amount of hUHRF1 in the cell lysate by doping in concentrated, purified, recombinant hUHRF1, confirmed the probe (9) could bind and photo-crosslink to hUHRF1 under these condi,ons (Figure and). Lastly, we wanted to ensure that even low levels of hUHRF1 were bound by our PI5P probe (9). To accomplish this, we expressed and purified FLAGtagged hUHRF1 and showed that the diazirine probe 9 could label it (Figure ). Then, lysate of cells expressing the FLAG-tagged hUHRF1 was treated with the probe (9), and by using an,-FLAG affinity chromatography, we demonstrated that we could isolate PI5P probe 9 labelled FLAG-hUHFR1, confirming the ability of the probe to bind even the low levels of expressed hUHRF1 without the need for doping (Figure ). Together these data support the u,lity of the diazirine probe (9) as a tool for finding and visualising PI5P binding partners.