AlphaLISA SureFire Biotin-Free Human and Mouse Total NEK7 Detection Kit, 10,000 Assay Points

RAW 264.7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂.Cells were treated with increasing concentrations of NK7-902 for 18 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NEK7 and ERK Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, treatment with NK7-902, a NEK7 molecular glue degrader, resulted in a dose-dependent decrease of NEK7 levels while Total ERK levels remained unchanged.

Specificity of NEK7 Total assay

Specificity of the NEK7 Total assay was assessed by assaying recombinant NEK6 and NEK7 proteins. Serial dilutions of recombinant proteins were prepared in Lysis Buffer and NEK7 levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of protein was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

NEK7 signal was only observed with NEK7 recombinant protein, no cross reactivity was detected with NEK6 recombinant protein. This result demonstrates the specificity of the Total NEK7 AlphaLISA SureFire Biotin Free assay as these two proteins share 81% sequence identity.

NEK7 expression in various cell lines

Adherent cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂.Suspension cells were harvested and seeded in a 96-well plate (400,000 cells/well).

Cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). NEK7 levels were evaluated using the AlphaLISA SureFire Ultra Biotin Free assay. For the detection step, 10 µL of cell lysate (4,000 adherent cells or 40,000 suspension cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, NEK7 protein expression was higher in Jurkat, NIH/3T3, and HeLa cells with low basal levels detected in MCF7 cells.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from Jurkat cells resuspended at 3 x 10⁶ cells/mL in HBSS. Cells were lysed with the addition of 5X Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and NEK7 levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. The assay can detect NEK7 expression in less than 500 cells.