AlphaLISA® SureFire® Ultra™

In association with PerkinElmer, TGR BioSciences is proud to announce the global release of its new flagship assay product, AlphaLISA® SureFire® Ultra™ to meet the demanding needs of researchers for phosphoprotein research.

AlphaLISA SureFire Ultra has been designed to provide exceptional assay sensitivity, and greatly enhanced signal windows for kinase screening programs. Measuring endogenous cellular phosphoproteins can now be achieved in all types of cellular extracts, including cultured cells, tissue lysates, and in samples containing high levels of antibodies. Such samples include those where antibody biotherapeutics are being tested for efficacy.

In addition, the assays have extremely low background signals, allowing testing of very low levels of pathway activation. Combined with the introduction of AlphaLISA technology, the assays provide unmatched performance for phosphoprotein detection.

AlphaLISA SureFire Ultra has been designed to provide exceptional assay sensitivity, and greatly enhanced signal windows for kinase screening programs.

First panel of AlphaLISA SureFire ULTRA targets shown in red.

Assay Principle

AlphaLISA® SureFire® Ultra™ assay kits allow the rapid, sensitive and quantitative detection of phosphoproteins from cells.  The AlphaLISA® SureFire® Ultra™ assay kits are optimized for enhanced signal-to-noise windows and compared to AlphaScreen® SureFire®, uses shorter incubation times and larger pipetting volumes.

The kits utilize Alpha beads that are each coated to specifically capture one of the assay antibodies. The Donor bead is coated with streptavidin to capture the biotinylated antibody while the Acceptor bead is coated with a proprietary “CaptSure™” agent that immobilizes the other antibody which is labeled with a CaptSure™ tag.  In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor beads in close proximity, enabling the generation of an Alpha signal upon illumination of Donor beads by an Alpha-enabled plate reader.  The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.

AlphaLISA® SureFire® Ultra™ assays utilize bead-based Alpha Technology and requires an Alpha Technology-compatible plate reader, such as PerkinElmer’s EnSight™ and EnSpire® Multimode and EnVision® Multilabel Plate Readers.  Alpha technology eliminates the need for laborious techniques, such as Western blotting or conventional ELISA. It is a homogeneous assay with no washing steps and this, combined with the short incubation times and excellent reproducibility (lower CV%) make, it highly amenable to robotic automation.

Assay principle

Example data:

  1. Measurement of p-AKT (Ser473) in the presence of extraneous antibodies – no interference of analyte detection with AlphaLISA SureFire Ultra by sample antibodies.
    image1
  2. Measurement of p-ERK in the presence of extraneous antibodies – no interference of analyte detection with AlphaLISA SureFire Ultra by sample antibodies.
    image2
  3. Measurement of inhibition of p70 S6 Kinase phosphorylation by AlphaLISA SureFire Ultra: comparison with SureFire.
    Note the dramatically increased signal-to-background window, and benefits for low-level detection.
    image3
  4. Multi-target analysis of cellular targets with AlphaLISA SureFire Ultra
    The ability to measure multiple different cellular targets on a single assay plate, with no wash steps, provides the potential for full automation of HTS pathway analysis. We show here the measurement of 7 phosphoprotein targets, and total proteins for two of these targets from individual culture wells of insulin-stimulated MCF-7 cells.
    image4

Kits are available worldwide from PerkinElmer.

Alphabetical list of kits available, with PerkinElmer website links:

p-JNK1/2/3 (Thr183/Tyr185)
p-MEK1 (Ser218/222)
p-Ribosomal Protein S6 (Ser240/244)
p-SLP-76 (Ser376)
p-STAT1 (Tyr701)
p-STAT1 (Ser727)
p-mTOR (Ser2448)
p-GSK-3b (Ser9)
p-STAT4 (Tyr693)
p-AKT1/2/3 (Thr308)
p-AKT1/2/3 (Ser473)
p-CREB (Ser133)
p-4EBP1 (Thr37/46)
p-EGF Receptor (Tyr1068)
p-eIF2α (Ser51)
p-eIF4E (Ser209)
p-ERK1/2 (Thr202/Tyr204)
p-IKKα (Ser176/180
p-IGF1 Receptor β (Tyr1135/1136)
p-Insulin Receptor β (Tyr1150/1151)
p-NFkB p65 (Ser536)
p-p38MAPK (Thr180/Tyr182)
p-p70S6K (Thr389)
p-SMAD1 (Ser463/465)
p-SMAD3 (Ser423/425)
p-STAT3 (Tyr705)
p-STAT5 (Tyr694/699)
p-VEGF Receptor 2 (Tyr1175)
Total STAT3
Total STAT5
Total MEK1
Total ERK1/2
Total AKT1
Total GAPDH
Total p38 MAPK

Manuals of the kits are available from the Perkin Elmer website.

For FAQ and troubleshooting information, click here.

For more information on SureFire, click here.

More information on the Alpha technology is available on the general PerkinElmer Technical Support pages: www.perkinelmer.com/ASK