Early Growth Response 1 (EGR1) is an immediate-early transcription factor that rapidly responds to diverse stimuli including growth factors, cytokines, and stress signals to regulate cell proliferation, differentiation, and apoptosis. EGR1 is induced within minutes of stimulation through MAPK and calcium signaling pathways, binding to GC-rich sequences in target gene promoters. EGR1 regulates expression of genes involved in cell cycle control (p53, PTEN), differentiation (fibronectin, PDGF), and vascular responses (VEGF, tissue factor). EGR1 functions as both a tumor suppressor and oncogene depending on cellular context, with loss of expression in prostate cancer and glioblastoma but overexpression in breast cancer. EGR1 plays critical roles in vascular injury responses, wound healing, and synaptic plasticity, making it relevant to cardiovascular disease, fibrosis, and neurological disorders.
The AlphaLISA SureFire Biotin-Free Human and Mouse Total EGR1 Detection Kit is a sandwich immunoassay for the quantitative detection of total EGR1 in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Biotin Free kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Biotin rich samples
AlphaLISA SureFire Biotin Free kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Total-AlphaLISA SureFire Biotin Free assay principle
The Total-AlphaLISA SureFire Biotin Free assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Biotin Free assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with a proprietary CaptSure3 agent to capture one of the detection antibodies, which is labeled with the CaptSure3 tag. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.
Total-AlphaLISA SureFire Biotin Free two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Biotin Free detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Total-AlphaLISA SureFire Biotin Free one-plate assay protocol
Detection of Total target protein with AlphaLISA SureFire Biotin Free reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire quality.
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.
