Interferon regulator factor 5, IRF5, is a transcriptional regulator of type I interferon (IFN-alpha and IFN-beta)-dependent immune responses. This transcription factor plays a key role in the innate immune response against DNA and RNA viruses. IRF5 is present in the cytoplasm of uninfected cells in an inactive form. Various mediators of the pathways that follow viral infection can overexpress and phosphorylate IRF5. IRF5 is associated with various cancers and autoimmune diseases.
The AlphaLISA SureFire Ultra Human and Mouse Total IRF5 Detection Kit is a sandwich immunoassay for the quantitative detection of total IRF5 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 Ultra kits are compatible with
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Alpha SureFire kits can be used for
Cellular kinase assays
Receptor activation studies
Screening
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Ultra 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 streptavidin to capture one of the detection antibodies, which is biotinylated. 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 Ultra 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 Ultra 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 Ultra one-plate assay protocol
Detection of Total target protein with AlphaLISA SureFire Ultra 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.
Validation of Phospho (Ser446)/Total IRF5 in CpG-B treated cells
RPMI 8226 cells were seeded in a 96-well plate (500,000 cells/well) in complete medium and incubated for approximately 10 minutes at 37°C, 5% CO2. The cells were treated with increasing concentrations of CpG-B, ODN-2006 or ODN-1668 (MedChem Express, HY-150218, HY-150726 respectively) for 6 hours.
After treatment, the cells were spun down at 1200 rpm for 5 minutes, washed with HBSS + 0.1 % BSA and then lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF5 Phospho (Ser446) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 50,000 cells for ODN-2006 experiment and 20,000 cells for ODN-1668 experiment) 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.
As expected, CpG-B triggered a dose-dependent increase in the levels Phospho (Ser446) while Total IRF5 levels remained unchanged.
Validation of Phospho (Ser446)/Total IRF5 in Calyculin A treated cells
THP-1 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated for approximately 10 minutes at 37°C, 5% CO2. The cells were treated with increasing concentrations of Calyculin A for 2.5 hours.
After treatment, the cells were spun down at 1200 rpm for 5 minutes, washed with HBSS + 0.1 % BSA 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 using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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.
As expected, Calyculin A triggered a dose-dependent increase in the levels IRF5 Phospho (Ser446) while Total IRF5 levels remained unchanged.
Specificity of IRF5 Total assay
Total IRF5 protein levels were assessed in A549 wild type (WT) and A549 IRF5 knockout (KO) (Abcam, ab301006) cells. Both cell lines were grown to confluency in a T75 flask in medium at 37°C, 5% CO2. Each flask was lysed in 2 mL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Lysates were then evaluated for Total IRF5 using the AlphaLISA SureFire Ultra kit.
For the detection step, 10 µL of cell lysate 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 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, Total IRF5 protein was detected only in A549 WT cells and no signal was detected in the KO IRF5 A549 cell line, demonstrating assay specificity.
Evaluation of IRF5 Total expression in Peripheral Blood Mononuclear Cells
Peripheral Blood Mononuclear Cells (PBMCs) were isolated from healthy donors using Ficoll® Plaque Plus (Merck GE17-1440-02).
Cells were seeded in 96-well plate (400,000 cells/well) and lysed in 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Generated lysate was then serially diluted in Lysis Buffer and evaluated using the AlphaLISA SureFire Ultra Total IRF5. For the detection step, 10 µL of cell lysate (starting from approximately 40,000 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. The number of cells/datapoint is indicated on graph.
Differential expression of IRF5 Total in various cell lines
Human and mouse cell lysates were diluted with Lysis Buffer. Approximate number of cells/datapoint is indicated on graph. Total IRF5 levels were evaluated using the AlphaLISA SureFira Ultra assay kit. For the detection step, 10 µL of cell lysate 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.
Total IRF5 expression varies depending upon cell type. As expected, a very high level of expression was detected in human cell lines: THP-1, KARPAS-299 and U937 and mouse cell lines: RAW 264.7 and L929.
