EGR1 Total Biotin Free Assay Kit Human and Mouse

The AlphaLISA™ SureFire® Ultra™ assay enables the rapid and sensitive detection of total and phosphorylated cellular proteins. AlphaLISA™ assays utilize two bead types: Acceptor Beads and Donor Beads. The Acceptor Bead is coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody which is labeled with a CaptSure™ tag. The Donor Bead is coated with streptavidin to capture the biotinylated antibody.  

In the presence of a target protein, the two target-specific antibodies bring Donor and Acceptor Beads into close proximity. When the Donor Beads are activated by a laser (680 nm), singlet oxygen is transferred to the Acceptor Bead leading to the production of an Alpha signal. The amount of light emission (615 nm) from the Acceptor Bead is directly proportional to the amount of target protein present in the sample. If an Acceptor Bead is not in close proximity (i.e. 200 nm) of a Donor bead, little to no signal is produced over background. 

The assay can be executed in a 1-plate or 2-plate assay protocol (Refer to Manual for more details).  

1-plate assay protocol: culturing of cells, treatment, lysis and assay are performed in a single well, enabling miniaturization in high throughput screening programs. 

2-plate assay protocol: cells are cultured and treated in a 96-well culture plate and lysates are then transferred into a separate plate for assay. This format allows the evaluation of multiple targets from a single lysate. 

Data obtained with a 2-plate, 2-incubation protocol. HeLa cells were seeded at 40K cells/well in a 96 well plate and incubated overnight. Cells were treated with EGF at the indicated concentrations for 2 hours. Cells were lysed with Lysis Buffer and assayed for Total EGR1 and ERK using respective SureFire® Biotin Free kits. Equivalent to approximately 4,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. Raw 264.7 cells were seeded at 40K cells/well in a 96 well plate and incubated overnight. Cells were treated with LPS at the indicated concentrations for 2 hours. Cells were lysed with Lysis Buffer and assayed for Total EGR1 and ERK using respective SureFire® Biotin Free kits. Equivalent to approximately 4,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. Jurkat cells were seeded at 400K cells/well in a 96 well plate in complete RPMI 1640 medium and treated with PMA at the indicated concentrations for 3 hours. Cells were lysed with the addition of 5X Lysis Buffer and assayed separately for Total EGR1 and ERK using respective SureFire® Biotin Free kits. Equivalent to approximately 16,000 cells/datapoint.

HeLa wild type (WT) and HeLa EGR1 knockout (KO, Abcam ab274929) cell lines were cultured to confluence in T175 flasks. Cells were treated with 2 ng/mL EGF for 2 hours, lysed in Lysis Buffer, serially diluted with Lysis Buffer, then assayed for Total EGR1 using the SureFire® Biotin Free kit. Approximate number cells/datapoint is indicated.

Data obtained from measurement of EGR1 Total in various cell types lysed with Lysis Buffer. Equivalent to approximately 5,000 cells/datapoint for adherent cells or 16,000 cells/datapoint for suspension cells.