Overview
Alpha no-wash assay technology (AlphaLISA™ and AlphaScreen® assays) is widely used in both basic research and in high throughput screening. The basic assay platform can be adapted to perform a wide variety of applications. These include:
- Immunoassays for analyte detection
- Protein-protein, protein-peptide, and protein-nucleic acid interaction assays
- Protein modification assays (phosphorylation, ubiquitination, histone methylation and acetylation, etc.)
Given the wide range of possible applications for Alpha assays, many different buffer types and compositions may be considered for use in Alpha assays. One of the Alpha immunoassay buffers supplied by Revvity may be ideal for your needs. Many other buffer compositions also can be used. This section will help you select a suitable buffer.
Avoiding assay interference from buffer components
Although Alpha technology is a robust platform that is tolerant of a wide range of conditions, some buffer components and solutions may lead to interference with the assay by various mechanisms. Here we describe the tolerance of Alpha technology to various buffer conditions and components. This information will help you to more quickly find optimal conditions for your assay. It is important to note that these guidelines refer to the Alpha assay chemistry itself (the Donor beads, Acceptor beads and chemistry that leads to light emission). Other buffer or sample components have the potential to affect the biology of the analyte or of analyte-specific antibodies that may be used in the Alpha assay.
Alpha-compatible Buffers Available from Revvity
Several pre-formulated buffers are available for Alpha assays. These have been developed to meet the varying needs of different Alpha assays, and one of them may be a good choice for an assay that you are developing. Their applications are compared in the table below.
| AlphaLISA buffer | Cat. No. | Comments |
|---|---|---|
| AlphaLISA immunoassay buffer | AL000 | A broadly applicable buffer,used in most AlphaLISA immunoassay kits. Contains detergent and low salt, so it can be used to lyse cells in some assays. Generally sufficient to expose the protein epitopes of membrane proteins for Alpha assay detection. |
| AlphaLISA universal buffer | AL001 | Used for quality control of all AlphaLISA and AlphaScreen Toolbox beads. It is universal since all QC assays will work in it, but it may not be the optimal buffer for all AlphaLISA assays. |
| AlphaLISA lysis buffer | AL003 | Contains SDS in order to lyse cells and solubilize proteins in cell-based assays. This buffer is required when detecting intracellular proteins. |
| AlphaLISA HiBlock buffer | AL004 | Used in situations where high background from sample components could interfere with the immunoasay. |
| AlphaLISA dissociation buffer | AL006 | Used to dissociate Protein A from IgG in bioprocess samples. Included in the AlphaLISA Residual Protein A kit (AL287) and available as a make-to-order product. |
| AlphaLISA NaCl buffer | AL007 | Contains 0.5M NaCl to help reduce background or interference in certain assays. Included in AlphaLISA mAlbumin kit (AL508) and m/r MCP1 kit (AL509), and available as a make-to-order product. |
| AlphaLISA Epigenetic buffer 1 | AL008 | Prevents the non-specific binding of the highly charged histone H3 (1-21) peptide to Donor and Acceptor beads in AlphaLISA epigenetic assays. Validated with Acceptor beads anti-H3K9ac (AL114), anti-H3K4me1-2 (AL116) and anti-H3K9me2 (AL117). It is used at the detection step for the dilution of Acceptor and Donor beads, not for the enzymatic reaction step. This buffer is not recommended for assays using full length histone proteins or nucleosomes. |
| Casein buffer | AL014 | Casein buffer for prevention of non-specific binding in AlphaLISA no-wash assays. This product is designed to be used in AlphaLISA binding kits. The Casein Buffer was a specially developed buffer to reduce the background in AlphaLISA binding assay. |
| PPI buffer | AL015 | General protein-protein interaction assay buffer for AlphaLISA no-wash binding assays. The PPI buffer (5X) has been specially developed for AlphaLISA protein-protein interaction assays. |
| MES buffer | AL016 | Low pH buffer for AlphaLISA protein-protein interaction assays that are pH-dependent. This buffer consists of 125 mM MES pH=6.0, 500 mM NaCl, 0.05% Tween 20, 0.5% Casein, 2.5% Triton X-100, 5 mg/mL Dextran and 0.25% ProClin-300. |
Buffering Agent
Alpha assay technology is tolerant to a wide range of buffering agents, buffer concentrations and pH ranges. The following buffering agents have been tested in Alpha assays and have been shown to give excellent performance at concentrations from 10 mM to 100 mM. Note that phosphate buffers should be generally avoided for assays measuring phosphorylation.
Alpha-compatible buffering agents (10 mM to 100 mM): Acetate, Bis-TRIS, Bis-TRIS propane, CAPS, carbonate, citrate, formate, HEPES, MES, MOPS, phosphate, PIPES and TRIS.
Buffer pH
Alpha technology is also very tolerant of assay pH. A range of pH from 2.5 to 9 has no influence on the performance of Alpha beads. Even pH up to 10.5 can be accommodated, but a slight loss of performance should be expected. The antibodies or other binding partners used in the Alpha assay may, of course, require a more limited pH range for optimal activity.
Buffer Salts
Buffers used in biological assays will usually include a variety of salts to generate ionic strength and to satisfy the specific requirements of an assay component such as an enzyme or protein. Alpha technology is highly tolerant of the presence of a wide variety of salts in solution. Alpha assays can tolerate the following ions up to 300 mM in solution.
Alpha-compatible ions (up to 300 mM):
Positive ions - Lithium (Li+), Sodium (Na+), Potassium (K+), Cesium (Cs+), Magnesium (Mg2+), Calcium (Ca2+), Ammonium (NH4+)
Negative ions - Fluoride (F-), Chloride (Cl-), Bromide (Br-), Iodide (I-), Borate, Acetate, Bicarbonate, Carbonate, Phosphate (both monobasic and dibasic), Sulfate, Pyrophosphate, Tartrate
Heavy metals must be considered carefully. Alpha assays have shown sensitivity to heavy metals in solution, most likely because those ions can react with singlet oxygen to form insoluble oxides. The following table describes the effects of commonly used heavy metals on Alpha assay performance:
| Metal ion | Recommended level | Interference level (IC50) |
|---|---|---|
| Cobalt (Co2+) | 0.7 mM or less | 3.6 mM |
| Iron (II) (Fe2+) | 0.2 mM or less | 0.95 mM |
| Iron (III) (Fe3+) | 2 mM or less | 9 mM |
Manganese (Mn2+) |
7 mM or less | 37 mM |
| Nickel (Ni2+) | 0.5 mM or less | 2.8 mM |
| Zinc (Zn2+) | 0.12 mM or less | 2.69 mM |
Due to their steep inhibition curves, a concentration of five times (5X) less than the IC50 can be considered as having no effect on the assay. The concentrations listed are the final concentrations in the presence of beads. The heavy metal concentration can be higher than the value shown at initial stages of the assay, so long as it is at or below the recommended level after addition of the final assay components.
Detergents and Background-reducing Proteins
Many assays will require the presence of additives to prevent aggregation, to ensure solubility and stability of components, and to reduce non-specific signal (background). Alpha technology can tolerate the presence of a wide variety of detergents and protein additives, but care must be taken in some cases.
Detergents
The following detergents will have no negative effect on Alpha assays at up to 3% final concentration in solution.
Alpha-compatible detergents (up to 3%):
Brij-35, Dodecane sulfonate, EMPIGEN, IGEPAL CA630, Mowiol 4-88, Nonidet P40, NSB195, NSB201, NSB256, Tergitol NP9, Triton X100, Tween 20, Tween 80, Dodecyltrimetylammonium bromide (DTAB), Octyl sulfate
Other detergents have to be considered carefully, since, at high concentration, they will have a significant effect on the technology. This effect is most likely due to denaturation of proteins such as streptavidin present on the surface of the beads. The following table illustrates those detergents and their effects. These recommended concentrations have been obtained by analysis of a dose-response curve using the TruHits kit.
| Detergent | Recommended level | Interference level(EC50) |
|---|---|---|
| BHDA (Benzylhexadecyldimethylammonium chloride) | 0.005% or less | 0.03% |
| Benzalkonium chloride | 0.005% or less | 0.03% |
| CHAPS | 0.02% or less | 0.10% |
| Chenodeoxycholate | 0.002% or less | 0.02% |
| Cholate | 0.002% or less | 0.15% |
| Decyl sulfate | 0.1% or less | 0.50% |
| Deoxycholate | 0.003% or less | 0.03% |
| myristyl sulfobetaine | 0.1% or less | 0.50% |
| Pluronic F127 | 0.1% or less | 0.30% |
| Sodium lauryl sulfate (SDS) | 0.02% or less | 0.20% |
Proteins and polymers
Proteins and other polymers are commonly added to reduce assay background. Alpha assays are tolerant of the following additives at up to 0.5% in solution.
Alpha-compatible proteins and polymers (up to 0.5%)
Bovine serum albumin (BSA), casein, gelatin, heparin, poly-lysine, salmon sperm DNA, Dextran T500 (both from natural and synthetic sources)
However, sources of BSA and casein have been found to vary in quality. These proteins must be of a high purity to avoid the presence of contaminants that could interfere with the assay. One such impurity is biotin, which is often present in casein of low grade. Other frequent contaminating proteins are immunoglobulins (IgGs) which can interfere depending on the assay configuration. We recommend the following grades for assays involving Alpha Technology.
- BSA (Sigma Cat.No. A7030)
- Casein 5% solution (Novagen Cat. No. 70955)
Anti-foaming agents
Finally, we observed an increasing popularity for anti-foaming agents in HTS assays. These molecules are designed to break the colloidal interactions between molecules of detergents and proteins such as BSA and thereby eliminate the presence of bubbles. Bubble formation can greatly hinder liquid handling and reading. The most common antifoam agents are Antifoam A (Cat. No. A5633) and Antifoam 204 (Cat. No. A8311) from Sigma. These agents show no interference with AlphaScreen assays at concentrations up to 0.1%, and Antifoam A was was more effective in reducing bubbles at that concentration than Antifoam 204.
Cofactors, Reducing Agents and Preservatives
Many assays require the presence of cofactors, reducing agents or preservatives in the assay buffer. AlphaScreen assays are tolerant of a wide variety of these molecules, including the following categories:
| Category | Alpha-compatible additives |
|---|---|
| Nucleotides, up to 10 mM | All nucleotides derived from adenine, guanine, thymine and uridine, including free nucleotide, mono-, di- and triphosphate and cyclic nucleotides |
| Reducing agents, up to 10 mM | Dithiothreitol (DTT), 2-mercaptoethanol, tris (2-carboxyethyl) phosphine (TCEP) |
| Preservatives, up to 0.1% | Kathon, thimerosal, ProClin 300 |
Note: Sodium azide is one of the most popular preservatives used with commercial products. However, it is known as a potent singlet oxygen quencher and thus it will quench Alpha signal strongly (IC50 of 0.005%). Sodium azide should be avoided in Alpha assays.
Enzymatic Inhibitors
Many assays require the addition of enzyme inhibitors, either to prevent degradation of assay components (protease, phosphatase) or to actually stop enzymatic activity as part of an assay (such as in kinase assays). Fortunately, Alpha technology is very tolerant of these compounds in solution.
| Category | Alpha-compatible additives |
|---|---|
| Protease inhibitors, up to 10 mM | AEBSF (and analogs such as PMSF), aprotinin, benzamidine, bestatin, E-64, leupeptin, N-ethylmaleimide, pepstatin |
| Protease inhibitors, up to 1 mM | antipain, chymostatin, phosphoramidon |
| Phosphatase inhibitors, up to 100 mM | sodium fluoride, sodium glycerophosphate |
| Phosphatase inhibitors, up to 10 mM | Sodium orthovanadate |
| Kinase inhibitors, up to 10 mM | magnesium chelators: EDTA, EGTA, 1,10-phenanthroline |
Bead-specific Buffer Interference Tables
View information for bead-specific interference. Our R&D team has tested a set of very common buffer components on each of our AlphaLISA toolbox bead products. Results are linked from this page.
Resources for Alpha Assay Optimization
- Assay Support Knowledge Base - Create your own Alpha assay
For research use only. Not for use in diagnostic procedures.