AlphaLISA SureFire Ultra Multiplex Biotin-Free Human Phospho-IRF5 (Ser446 & total) Detection Kit, 10,000 Assay Points

Multiplex SureFire Ultra Biotin Free assay principle

The Multiplex SureFire Ultra Biotin Free assay simultaneously measures phospho and total protein at a specific level in a biological sample (e.g. cell lysate). It utilizes three antibodies that recognize the phospho epitope and two distal epitopes on the target protein. The assay requires three bead types: two distinct Acceptor Beads and one Donor Bead.

• The Alpha 615 Acceptor Bead is coated with the CaptSure™ antibody, which binds the CaptSure-tagged anti-phospho target antibody.
• The Alpha 545 Acceptor Bead is coated with the CaptSure2 antibody, which binds the CaptSure2-tagged anti-total target protein antibody.
• The Alpha Donor Bead binds the CaptSure3 anti-total target protein antibody.

When the phosphorylated protein is present, the three antibodies bring the Donor and Acceptor Beads close together, allowing singlet oxygen to transfer energy and excite both Acceptor Beads. This generates a luminescent Alpha signal at 615nm and 545nm. The 615nm (Eu) signal is directly proportional to the amount of phosphorylated protein, while the 545nm (Tb) signal corresponds to the total protein levels.

Multiplex SureFire Ultra 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 Multiplex SureFire Ultra 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 different targets.

Multiplex SureFire Ultra Biotin Free one-plate assay protocol

Detection of Phosphorylated/Total target proteins with Multiplex SureFire Ultra 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 Ultra quality.

R848 mediated IRF5 activation

RPMI 8226 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium at 37°C, 5% CO₂. Cells were treated with 20 µM of TLR7/8 agonist, R848 for 2 hours.

After treatment, the cells were spun down and lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF5 Phospho (Ser446) and Total levels were evaluated in the same wells of an assay plate using the AlphaLISA SureFire Multiplex Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 40,000 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 AlphaPlex settings.

As expected, treatment with R848 increased phosphorylation of IRF5 at Ser446 with no significant change to Total levels.

LPS mediated IRF5 phosphorylation in RPMI 8226 cells

RPMI 8226 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium at 37°C, 5% CO₂. Cells were treated with increasing concentrations of LPS for 4 hours.

After treatment, the cells were spun down and lysed with 65 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF5 Phospho (Ser446) and Total levels were evaluated in the same wells of an assay plate using the AlphaLISA SureFire Multiplex Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 30,000 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 AlphaPlex settings.

As expected, treatment with LPS increased phosphorylation of IRF5 at Ser446 with no change to Total levels.

Increased IRF5 phosphorylation in response to CpG-B treatment

RPMI 8226 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium at 37°C, 5% CO₂. Cells were treated with increasing concentrations of CpG-B ODN 2006 for 2 hours.

After treatment, the cells were spun down and lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF5 Phospho (Ser446) and Total levels were evaluated in the same wells of an assay plate using the AlphaLISA SureFire Multiplex Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 40,000 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 AlphaPlex settings.

As expected, treatment with CpG-B increased phosphorylation of IRF5 at Ser446 with no change to Total levels.

Increased IRF5 phosphorylation with Calyculin A

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium at 37°C, 5% CO₂ and treated with increasing concentrations of Calyculin A for 3 hours.

After treatment, the cells were spun down and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF5 Phospho (Ser446) and Total levels were evaluated in the same wells of an assay plate using the AlphaLISA SureFire Multiplex Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 20,000 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 AlphaPlex settings.

As expected, treatment with Calyculin A increased IRF5 phosphorylation at Ser446 with no change to Total levels.

Differential expression of IRF5

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 200,000 cells/mL. Suspension cells were harvested and lysed with Lysis Buffer at 200,000 cells/mL.

IRF5 Total levels were evaluated using the AlphaLISA SureFire Multiplex Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 20,000 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 AlphaPlex settings.

Total IRF5 expression varies depending upon cell type. High levels of expression was detected in THP-1, U937 and RAW 264.7.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from THP-1 cells treated with 100 nM Calyculin A for 3 hours at 37°C, 5% CO₂ and lysed at a final density of 8 x 10⁶ cells/mL.

Lysates were serially diluted in Lysis Buffer and assayed for Phospho (Ser446) and Total IRF5 levels using the AlphaLISA SureFire Multiplex Biotin Free kit. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL 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 AlphaPlex settings.

Approximate number of cells/datapoint is indicated on the graph. Assay background is represented by the dotted line.