Alpha SureFire® Ultra™ Multiplex

In association with PerkinElmer, TGR BioSciences is proud to announce the global release of its next-generation assay product, Alpha SureFire® Ultra™ Multiplex, the first homogeneous multiplexing assay kit for phosphoprotein research.

Alpha SureFire® Ultra™ Multiplex allows the simultaneous detection of two targets in a single well, with the benefits of high sensitivity, and no washing steps. The system utilizes TGR’s CaptSure™ technology to provide exceptional assay sensitivity, as well as reduced sample interference.

The first products on-market are phospho/total assay kits. Measuring endogenous cellular phosphoproteins plus the total levels 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.

The Alpha SureFire® Ultra™ Multiplex can also be used to measure two phosphoproteins at the same time, and further products for this application will be available soon.

Measurement of phospho and total protein targets:

Measurement of phospho and total protein targets

Measurement of two phosphoprotein targets:

Measurement of two phosphoprotein targets

Assay Principle

The Alpha SureFire® Ultra™ Multiplex Phospho + Total assay kits and Phospho + Phospho assay kits (to be released soon) allow the rapid, sensitive, and quantitative detection of phosphoproteins from cells, combined with the measurement of the total amount of the same protein or a second phosphoprotein.  This Alpha Multiplex measurement is carried out in the same assay plate well from a single sample of cell lysate, and is achieved by the use of two types of Alpha Acceptor beads that emit at distinct wavelengths (545nm and 615nm).

The two distinct Alpha Acceptor beads report their binding to a distinct antigen through their association with specific assay antibodies, as indicated below.

Single target – Phospho + Total Assay kits

The Alpha 615 Acceptor bead is directly-conjugated with an antibody to the phosphorylated site on the target protein. The Alpha 545 Acceptor bead is coated with the CaptSure™ agent, which binds the CaptSure tagged anti-total target protein antibody. The Alpha Donor bead binds the biotinylated anti-total target protein antibody.

Dual target Assay kits

The Alpha 615 Acceptor bead is directly-conjugated with an antibody to the phosphorylated site on the first target protein. The Alpha 545 Acceptor bead is coated with the CaptSure agent, which binds the CaptSure tagged anti-phospho antibody of the second target protein. The Alpha Donor bead binds the biotinylated anti-total antibodies to both target proteins.

In the presence of target protein, the 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 target protein present in the sample.

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.

The assay utilizes the bead-based Alpha Technology, and requires an Alpha Technology-compatible plate reader capable of reading dual emission wavelengths. Click here for more information about the AlphaPlex technology and download the “AlphaPlex Quick Start Guide” and the “AlphaPlex Assay Development Guide” to find guidance about filters and mirrors selection, instrument protocol and channels crosstalk correction.

Example data

Simultaneous measurement of p-AKT (Ser473) and Total AKT in each well

MCF-7 cells were plated overnight at 200K/mL in 200 mL MEM + 10% FCS. Cells were then treated for 2 hours with varying concentrations of wortmannin in MEM +1% FBS, and then stimulated for 30 min with 2.5mg/mL insulin (n = 3 for each condition). Each well was lysed in 100 mL SureFire Ultra lysis buffer, and 10 µL aliquots of each lysate were assayed simultaneously for p-AKT 1/2/3 (Ser473) and total AKT1 using the Alpha SureFire Ultra Multiplex kit.

Simultaneous measurement of p-AKT (Ser473) and Total AKT in each well

Simultaneous measurement of p-ERK and Total ERK in each well

A431 cells were plated overnight at 200K/mL in 200 mL MEM + 10% FCS. Cells were then serum starved for 2 hours, and then stimulated for 10 min with various concentrations of EGF (n = 3 for each condition). Each well was lysed in 100 mL SureFire Ultra lysis buffer, and 10 µL aliquots of each lysate were assayed simultaneously for p-ERK and total ERK using the Alpha SureFire Ultra Multiplex kit.

Simultaneous measurement of p-ERK and Total ERK in each well

Kits are available worldwide from PerkinElmer (www.perkinelmer.com).

Currently available:

p-ERK 1/2 (Thr202/Tyr204) 615 (Eu) + Total ERK 545 (Tb)

Cat #s: MPSU-PTERK-K500/K10K/K50K

 

p-Akt 1/2/3 (Ser473) 615 (Eu) + Total Akt 1 545 (Tb)

Cat #s: MPSU -PTAKT-K500/K10K/K50K

 

p-AKT1/2/3 (Ser473) + p-ERK1/2 (Thr202/Tyr204)

Cat #s: MPSU-PAKER-K500/K10K/K50K

 

Dual measurements of p-ERK1/2 (Thr202/Tyr204) or p-AKT1/2/3 (Ser473)
Multiplexed with any other SureFire Ultra target

Cat #s:

  • MPSU-PERK-E500/E10K/E50K
  • MPSU-PAKT-E500/E10K/E50K