AlphaLISA SureFire Biotin-Free Human and Mouse Total EGR1 Detection Kit, 50,000 Assay Points

Induction of EGR1 in cells treated with EGF

HeLa 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 EGF for 2 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). EGR1 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, EGF triggered a dose-dependent increase in the levels of Total EGR1 while ERK Total levels remained unchanged.

Induction of EGR1 in cells treated with LPS

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 LPS for 2 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). EGR1 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, LPS triggered a dose-dependent increase in the levels of Total EGR1 while ERK Total remained unchanged.

Induction of EGR1 in cells treated with PMA

Jurkat cells were seeded in a 96-well plate (400,000 cells/well) in complete RPMI 1640 medium and treated with increasing concentrations of PMA for 3 hours.

After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). EGR1 and ERK Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 16,000 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, PMA triggered a dose-dependent increase in the levels of Total EGR1 while Total ERK remained unchanged.

Knockout validation of EGR1 Total Assay

EGR1 levels were assessed in HeLa wild type (WT) and EGR1 KO (Abcam, ab274929) cell lines cultured to confluency in T175 flasks and treated with 2 ng/mL EGF for 2 hours. Each flask was lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking. Lysates were serially diluted in Lysis Buffer and levels of EGR1 Total were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate was transferred into a 384-well white OptiPlate, followed by 5 µL Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was 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.

EGR1 was only detected in WT cells, demonstrating assay specificity. Dotted line represents assay background.

EGR1 expression in various cell lines

Adherent cells were grown to confluency in a T175 flask at 37°C, 5% CO₂ and were lysed with Lysis Buffer at a density of 0.5 x 10⁶ cells/mL. Suspension cells were harvested, washed in HBSS and lysed with Lysis Buffer at 1.6 x 10⁶ cells/mL.

EGR1 levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (5,000 adherent and 16,000 suspension cells) were 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.