Overview

FlashPlates® are special plates that contain scintillant embedded into the plastic of the plate. In a FlashPlate format, cell membranes are captured onto the bottom and sides of the well of a FlashPlate. When the GPCR is activated, ³⁵S-labeled GTP will bind to the cell membrane. This puts the radiochemical into proximity of the bottom and sides of the FlashPlate. When the radiochemical is close to the edges of the well, the beta-energy from the ³⁵S can interact with scintillant embedded in the plastic of the well, producing signal. Unbound ³⁵S GTP will be floating freely in solution, and will not be close enough to the edges of the well to produce signal.

FlashPlate GTP binding assay

There are two different ways to capture cell membrane to the wells of the FlashPlate. You can use our Basic FlashPlates (uncoated), and directly-coat your cell membranes to the well. You can also use our WGA-coated FlashPlate to capture cell membranes. Wheat Germ Agglutinin (WGA) FlashPlates are a practical, easy-to-use, 96- or 384-well platform for high-throughput G Protein-Coupled Receptor (GPCR) binding assays. This technology incorporates proprietary covalent attachment of WGA to the microplate, providing a stable, high capacity binding capability. The interior of each FlashPlate well is permanently coated with a thin layer of polystyrene-based scintillant. The FlashPlate is then coated with proprietary proteins followed by Wheat Germ Agglutinin. The WGA captures receptors with carbohydrate moieties, thereby providing a platform for homogeneous, high-throughput receptor-ligand binding assays. GPCR occupation by agonists leads to guanine nucleotide exchange. GDP bound to Gα, of the Gαβγ complex, dissociates and is replaced by ³⁵S-gamma-GTP.

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What do I need to run this assay?

• Basic FlashPlate or WGA-coated FlashPlate (refer to table below)
• Cell membrane expressing receptor of interest. (爱游戏平台注册登录 carries receptor-transfected cell membranes)
• ³⁵S GTPγS (#NEG030H or NEG030X; refer to table below)
• Unlabeled non-hydrolyzable GTP-gamma-S for non-specific binding control
• GDP, agonists, antagonists, and test compounds as appropriate
• TopSeal-A™ (adhesive plate seal, Cat. No. 6050185)
• High throughput radiometric detector. (We recommend a TopCount® counter or MicroBeta® counter.)

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Products and catalog numbers

³⁵S gamma GTP radiochemicals

爱游戏平台注册登录 /New England Nuclear offers two different ³⁵S gamma GTP products in various sizes:

FlashPlates

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Protocols-in-brief

View a detailed sample protocol for a 5HT1A receptor FlashPlate GTP binding assay using directly-coated cell membranes.

Workflow for FlashPlate GTP binding assay

Example: CHO cells expressing the h5-HT1B receptor were diluted to a desired membrane protein concentration in 25 mM HEPES buffer containing 2.5 mM CaCl₂ and 1 mM MgCl₂, at pH 7.4. A suspension of diluted receptor (230 μl) was pipetted into each well of a FlashPlate microplate, and the plates were centrifuged at 800 x g for 10 minutes at 4°C. The supernatant was removed and the plates were left overnight at 4°C. (Later studies have shown that 3 hours is sufficient.) Prior to use, 250 μl of 0.5% BSA in the above HEPES buffer was added to each well and left for 30 minutes at room temperature before aspirating and letting plate dry, in order to block nonspecific binding sites.

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Assay optimizations

1. Cell membrane titration. (For WGA-coated FlashPlates, we recommend 1-10 μg/well.)
2. Concentration of GDP, MgCl2, NaCl, saponin in assay. We recommend testing 0-10 μM GDP, 1-10 mM MgCl2, 0-100 mM NaCl, and 3- 100 μg/mL saponin.
3. Incubation time. In one application note below that used directly-coated FlashPlates, the reaction was stopped after 30 minutes by aspirating and immediately read. In another application note, the reaction was incubated 4 hours prior to reading the WGA-coated plate (no aspiration).
4. Agonist dose-response curve and other assay validation assays (Z' experiment if applicable, etc.)

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Application note, posters, guides and other resources

• Application Note - compares the FlashPlate GTP binding assay to a filtration GTP binding assay, using membranes from CHO cells expressing the 5-HT1b receptor. Also has information on how to coat a Basic FlashPlate (uncoated FlashPlate) with cell membranes.
• Application Note - describes the use of a couple of our opioid receptor membranes in WGA FlashPlate and filtration GTP binding assays
• NIH assay guidance website for GTP binding assays
• FlashPlate technology knowledge base page

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Tips and FAQs

• The GTPγS assay works best with Gi-coupled GPCRs. Very low assay windows are usually obtained for Gs- and Gq-coupled receptors, due to both (1) levels of expression of Gi relative to Gs or Gq proteins and (2) the exchange rate of these G proteins for GTP.
• The GTPγS assay is sensitive to GDP concentration, concentration of ³⁵S GTPγS and concentration of Mg²⁺ in the assay buffer.
• Controls: we recommend that when you develop your assay, you include a no-agonist control (substituting with buffer) to determine your basal GTP binding level, and a non-specific binding (NSB) control where you use unlabeled non-hydrolyzable GTP with ³⁵S-gamma-GTP and cell membrane
• It is recommended to deliver the droplets in the center of the well. Pipetting along the side of the well can result in a phenomenon known as “tracking” when working with small volumes. What occurs is that the droplet sticks to the side of the well, which results in greater variability, as this material may not mix completely with the remainder of the assay components. This will cause a decrease in counts in the case of radiochemical addition or a compound showing less activity in the case of compound addition.
• If you are directly-coating your cell membranes to the FlashPlate, be careful not to scratch the bottom of the well while pipetting since that is the area where the membranes are coated. Disturbing the membranes will cause a loss of counts.
• Make sure that tips are washed after the addition of compounds. If tips are not washed there is a risk of contaminating the compound and assay plates due to carry-over.
• After the addition of the radioligand, cover the plates and incubate them in an area with no, or very little, light. If plates are exposed to light, excitation of the scintillant might occur. This would result in an increase in background noise and lead to deterioration in the quality of the obtained results.
• Do not shake or spin the plates during assay incubation. If you spin or shake your plates you may lose the liquid that is situated at the bottom of the FlashPlate® well. Therefore, it is recommended to gently mix the liquid by placing the FlashPlate®, which is covered by a TopSeal™, on a flat surface and moving it in circular motions (two turns would suffice). If you are working with a large number of FlashPlate® microplates, stack them on top of one another and then gently move them in a circular motion.
• It is important to normalize instruments with multiple detectors. If the instrument is not normalized then you will observe more variation in the readings. Each detector must be normalized to read optimally.
• It is important to perform a background subtraction on the instrument the first time it is set up for a FlashPlate assay. When setting up the FlashPlate assay for the first time it is recommended to perform a background subtraction on the instrument. This can be done with a plate of the same type to be used in the assay in the absence of radioactivity. The background counts in the assay will be lower and better S/B values will be obtained.
• Be careful of static when working in an extremely dry environment. Static will cause a peculiar jump in counts (spike). For example, 10,000 cpm are obtained whereas 300-400 cpm were expected. To remedy this problem one should avoid wearing large woolly sweaters. If this problem occurs, re-read the plate or swipe the bottom or side of the plate with a damp kim-wipe and re-read the plates.

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Data analysis

Visit our GTP binding data analysis page.

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Citations

1. Cao, X. et al. Blockade of cannabinoid type 1 receptors augments the antiparkinsonian action of levodopa without affecting dyskinesias in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys. J Pharmacol Exp Ther 323, 318-26 (2007). Link
2. Gaibelet, G. et al. Cholesterol content drives distinct pharmacological behaviours of micro-opioid receptor in different microdomains of the CHO plasma membrane. Mol. Membr. Biol 25, 423-435 (2008). Link
3. Wan, Y. et al. Identification of full, partial and inverse CC chemokine receptor 3 agonists using 35S GTP-gamma-S binding. Eur. J. Pharmacol 456, 1-10 (2002). Link
4. Watson, J., Selkirk, J.V. & Brown, A.M. Development of Flashplate Technology to Measure 35S GTPyS Binding to Chinese Hamster Ovary Cell Membranes Expressing the Cloned Human 5-HTIB Receptor. J Biomol Screen 3, 101-105 (1998). Link

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Custom cell lines, membranes, frozen cells, and receptors

爱游戏平台注册登录 offers custom cell lines and membranes as well as custom assay development. If you are interested in a custom receptor membrane, please contact our custom teams:

ON>POINT® Custom Assay Development Services

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