Working with cell extracts and supernatants in Alpha assays

Overview

One major advantage of the Alpha technology is that it is a homogeneous (no-wash) assay that can be used in many cell-based applications. Samples such as tissue homogenates, cell lysates, cell culture supernatants, and cell membranes have been used in Alpha assays.

When possible, we recommend aspirating the cell culture medium and performing your assay in a different reaction buffer or lysis buffer. If you are designing your own assay, the reaction buffer or lysis buffer should be optimized for your particular application and sample. If you need to perform your bead reaction in the culture medium, the AlphaLISA® Acceptor bead would generally be recommended over the AlphaScreen® Acceptor bead, as it offers reduced interference from phenol red and other components in complex matrices. You may still, however, want to evaluate culture medium that does not contain phenol red, to see how this impacts your assay performance. You also may want to titrate down the amount of FBS or other serums in your medium, to see how this affects your assay. RPMI media contains biotin, which can interfere to a small extent with Alpha assays. See the FAQs below for tips on using RPMI media.

When working with cells, you have the option of a 1-plate (all-in-one-well) or a 2-plate protocol. In a 1-plate protocol, the cells are treated in the same plate in which your assay will be read. In a 2-plate protocol, your sample is treated first in one plate, then the entire sample or a portion of the sample is transferred to another plate for the bead addition and incubation steps. Each format offers advantages. For example, a 1-plate assay usually requires fewer addition steps, which can be advantageous in high-throughput screening. A 1-plate assay also ensures that you are not losing any sample during a transfer step. A 2-plate protocol allows you to use portions of the same sample in different assays. A 2-plate protocol also allows you to work in a larger volume or higher concentration reaction during your treatment step before transferring just a portion of your sample to another plate for bead addition. The 2-step format also reduces the concentration of potentially interfering substances that may be present in the cell lysate or supernatant. You will want to consider the format that will work best for your needs.

If you are running an Alpha SureFire® assay, please refer to their respective application pages for information specific to these kits. These kits come with their own lysis buffers or lysis buffer recipes and instructions specific for their applications.

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Application notes and Posters

  • Application note: Protocol Optimization for Detection and Quantification of Membrane-Bound Proteins Using AlphaLISA Technology
  • Poster describing cell-based cytokine detection assays in AlphaLISA all-in-one-well format.
  • Poster describing a cell-based kinase assay in AlphaScreen format.

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Citations

View a brief list of citations where cell lysates or supernatants were used in an Alpha assay.

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Tips and FAQs

If you are running an Alpha cAMP or Alpha SureFire® assay, please refer to their respective application pages for information specific to these kits.

  • 1-plate assay: An assay where cells are treated and lysed in the same plate to which Donor and Acceptor beads will be added. You will not be transferring your cell lysate to a separate plate. This may be preferable when automating the assay for high throughput analysis.
  • 2-plate assay: An assay where cells are treated and lysed in one plate, and then the lysate is transferred to a second plate for addition of the beads.


Q. How do I lyse my cells?
A. The AlphaLISA ImmunoAssay buffer (catalog number AL000) is not per se a lysis buffer, but it does contain detergent and low salt, so it can be used to lyse cells in some assays. For membrane proteins, it has been shown that the AlphaLISA ImmunoAssay buffer is sufficient to expose the protein epitopes for AlphaLISA detection. Our AlphaLISA lysis buffer (catalog number AL003) was developed specifically for use in cell-based assays. This buffer contains SDS in order to lyse the cells and solubilize the proteins. This buffer is required when detecting intracellular proteins. Alternatively, we recommend a cell extraction buffer from BioSource (catalog number FNN0011). The recipe for this buffer is available online and can be made in-house: 10 mM Tris, pH 7.4 final, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, and 0.5% deoxycholate. Lysis buffers may need to be optimized for your particular application and sample.


Q. What is the difference between the various AlphaLISA buffers?
A. The AlphaLISA ImmunoAssay buffer (catalog number AL000) is not per se a lysis buffer, but it does contain detergent and low salt, so it can be used to lyse cells in some assays. For membrane proteins, it has been shown that the AlphaLISA ImmunoAssay buffer is sufficient to expose the protein epitopes for AlphaLISA detection. AlphaLISA HiBlock buffer (catalog number AL004) contains extra immunological blocking agents (compared with AlphaLISA ImmunoAssay buffer), and can be useful in cases where high background is a concern. The formulation of both these buffers is listed on the product technical data sheets. Our AlphaLISA Lysis buffer (catalog number AL003) was developed specifically for use in cell-based assays. This buffer contains SDS in order to lyse cells and solubilize proteins. This buffer is required when detecting intracellular proteins. AlphaLISA Universal buffer is the buffer used to QC all AlphaLISA and AlphaScreen Toolbox beads. AlphaLISA Universal buffer is universal since all QC assays will work in it, but it may not be the optimal buffer for all AlphaLISA assays.

Visit Buffer selection for Alpha assays for more help in choosing a suitable buffer for your assay. This page describes the various Alpha immunoassay buffers supplied by 爱游戏平台注册登录 and will help you avoid interfering substances in buffers of your own design.


Q. Can I work with tissues?
A. If you are working with tissues, you can try using 5% TCA to homogenize your sample. There is no interference from the TCA salt on the Alpha assay. However, you may want to be careful regarding how TCA will alter the pH (make sure your final sample solution is well-buffered). pH can affect the interaction of antibodies with binding partners, as well as other biomolecular interactions.


Q. My culture medium contains 10% FBS. Will this interfere?
A. Depending on your assay design, your sample will likely be diluted when mixed with beads, making the final concentration of FBS in the well lower than 10%. If your culture medium remains in the well when the assay is being read, you may want to titrate the amount of FBS in your culture media to see if this affects your assay performance. Please note that FBS might have an assay-dependent effect.

If the final concentration of FBS in your assay is 10%, this can quench the signal (possibly due to iron in the FBS - iron is a singlet oxygen quencher). You would most-likely need to titrate down the amount of FBS in your media.


Q. My culture medium contains phenol red. Will this interfere?
A. Phenol red can interfere with an AlphaScreen assay. If working with an AlphaScreen Acceptor bead, you should use a phenol red-free medium. If your culture medium (containing phenol red) is going to remain in the well when the assay is being read, you should use AlphaLISA Acceptor beads in your assay design. If you are working with a culture medium that contains higher amounts of phenol red than normal (for example, some formulations of DMEM contain 40 µM phenol red, which is rather high), you may need to switch to a phenol red-free medium or a culture medium formulation with less phenol red.


Q. I'm working with RPMI. Will this interfere?
A. RPMI medium can be used for Alpha assays. However, due to the presence of a high level of free biotin, a decrease in total counts and signal-to-background ratio (S:B) can be expected if you are using streptavidin-coated beads. Also, the presence of free biotin might affect the LDL (lower detection limit). In most cases, satisfactory results can still be obtained using RPMI medium. Increasing the concentration or incubation time with the streptavidin-coated Donor beads can help to overcome the presence of biotin in RPMI medium in some cases. Other cell culture media that work well in Alpha assays include DMEM and MEM.


Q. Can I bind a cell membrane to a bead? If so, what is the best way to do this?
A. You will want to test this for your particular assay. We have done this in the past with lectin-coated beads (wheat germ agglutinin, concanavalin A, etc.). We can custom conjugate beads to be coated with WGA and ConA, etc.


Q. Can I bind tissues or crude membrane preparations to the beads?
A. You will want to test this for your particular assay. We have used lectin-coated beads in the past to capture membrane. You may also be able to tag or biotinylate your membrane and use an appropriate affinity bead to capture your tissue.

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Custom bead conjugation and custom assay development at 爱游戏平台注册登录

爱游戏平台注册登录 offers custom bead conjugation services as well as custom assay development. If you are interested in having your biomolecule custom-conjugated to a bead, or in custom assay development, please contact our custom teams:

ON>POINT® Custom Labeling and Conjugation Services
ON>POINT® Custom Assay Development Services

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