Thermo Scientific™ LanthaScreen™ LRRK2 G2019S [pSer935] Cellular Assay Kit

Catalog No.	Quantity
A14686	1 Kit

Catalog No. A14686 Supplier Thermo Scientific™ Supplier No. A14686

Description

The LanthaScreen™ LRRK2 G2019S [pSer935] Cellular Assay Kit combines the flexibility of the BacMam gene delivery system with the robustness and power of our LanthaScreen™ TR-FRET technology.

The LanthaScreen™ LRRK2 G2019S [pSer935] Cellular Assay Kit combines the flexibility of the BacMam gene delivery system with the robustness and power of our LanthaScreen™ TR-FRET technology. It allows researchers to interrogate phosphorylation at serine 935 on a disease-relevant mutant (G2019S) Leucine-Rich Repeat Kinase-2 (LRRK2) protein in a variety of cell backgrounds to identify LRRK2 kinase inhibitors.

This assay format provides a convenient tool for the expression of G2019S LRRK2-GFP fusion protein in the cell background of your choice. The resulting cellular assay can be used to identify LRRK2 G2019S inhibitors, aiding in Parkinson's disease and other research focuses.

This kit provides enough material to evaluate the technology in a 384-well plate; larger sizes of all reagents are available separately.

The kit includes:

• LanthaScreen™ Tb-anti-LRRK2 [pSer935] Antibody, 5 μg
• BacMam LRRK2-GFP G2019S Reagent, 5 x 1 mL
• LanthaScreen™ 6X Lysis Buffer, 6 mL
• Instrument Control Terbium TR-FRET kit

A kit for interrogating wild-type LRRK2 protein (Cat. No. A14685) is also available.

Benefits of this assay include:

• More Physiologically Relevant: cell-based LRRK2 kinase activity and choice of cell background
• More Convenient: compatible with High Throughput Screening (HTS); LanthaScreen™ technology reduces interference and noise
• Disease Relevant: screen compound libraries for Parkinson's disease-relevant molecules

More Physiologically Relevant

The LanthaScreen™ Tb-Anti-LRRK2 [pSer935] Antibody allows the investigation of phosphorylation at serine 935 on LRRK2 protein kinase. When paired with LRRK2 BacMam reagents, you have the freedom to choose the cellular background for your assay, including primary cells. This enables screening for potential inhibitors of LRRK2 proteins in their natural complexes in a physiologically relevant cell type.

More Convenient

Assays are run in a fully homogenous, addition-only format without any of the washing, lysate transfer, or separation procedures required for traditional methods such as western blotting and ELISA, making this assay ideal for HTS applications. The LanthaScreen™ technology provides all of the advantages of TR-FRET ratiometric detection, including reduced data noise, less interference from fluorescent compounds, and high sensitivity, which results in the use of fewer cells than traditional Western or ELISA methods.

Disease Relevant

The LanthaScreen™ Tb-Anti-LRRK2 [pSer935] Antibody can be used by Parkinson's and other disease researchers to perform HTS, cell-based assays to generate inhibition curves (IC50) for their compound libraries. This will aid drug discovery efforts by reducing the time to results from 4 hrs to typically 1.5 hrs compared to alternative ELISAs.

Specifications

• Assay: Cell-Based Assay
• Assay Entry: Cell-based terbium modification activity (transient)
• Cell State: Cells Not Included
• Content And Storage: • BacMam LRRK2-GFP G2019S, 5 x 1 mL (Store at 4°C)
  • LanthaScreen™ 6X Lysis Buffer, 1 tube (Store at 4 °C)
  • LanthaScreen™ Tb-anti-LRRK2 [pSer935] Antibody, 1 tube (Store at -20°C)
  • Instrument Control Tb TR-FRET Kit (Store at 4°C)
  • LanthaScreen™ LRRK2 [pSer935] G2019S Cellular Asasy Kit Protocol (Store at room temperature)
• Detection Method: Fluorescence
• For Use With (Application): TR-FRET
• For Use With (Equipment): Microplate Reader
• Product Line: LanthaScreen
• Quantity: 1 Kit
• Readout: TR-FRET (Activity)
• Reporter Gene: GFP (EmGFP)
• Target Entry: LRRK2 G2019S
• Type: Cellular Assay Kit
• System Type: LanthaScreen™
• Druggable Target: Kinases

Frequently Asked Questions (FAQs)

How does the LanthaScreen technology compare to other TR-FRET assay formats?

We performed a comparison between the LanthaScreen assay and other commercially available TR-FRET assays from 2 different suppliers for the PKC kinase target. Our data revealed that the assays performed comparably, but that the LanthaScreen assay was simpler to optimize and contained fewer components that required optimization. The LanthaScreen assay is a two component system, whereas the other assay formats utilize a trimolecular mechanism which is more time consuming to optimize and has added costs.

For my kinase assay, can I pre-mix the Tb-Ab and EDTA so that I can stop the kinase assay and begin detection with a single reagent addition?

Yes, this is possible depending on the concentrations of reagents used and the time for which they are mixed. We recommend developing and optimizing the assay by using separate reagent additions, because this method will work under the widest range of conditions. Once the assay is optimized, the performance of the assay using pre-mixed antibody and EDTA can be evaluated. We have successfully developed robust assays in which the antibody and EDTA were pre-mixed and then stored overnight at 4 degrees C prior to use the following day. A loss of signal intensity was observed in this case, however, by using the ratiometric readout, this effect was minimal.

Are the LanthaScreen reagents stable to interference from Mg2+, Mn2+, and EDTA?

The chelate is completely stable to Mg2+. The amount of Mn2+ or EDTA that the chelate can tolerate depends largely on how long they are mixed together and the combination of additives used in the reaction. If a reaction requires either Mg2+ or Mn2+ for activation, it is best to stop the reaction by adding an equimolar amount (or slight excess) of EDTA to chelate the metal ions present. This will then essentially eliminate any interference on the terbium chelate by EDTA or Mn2+. Regardless, when LanthaScreen assays are performed using a ratiometric readout (division of the acceptor signal by the donor signal), any interference caused by Mn2+ or EDTA is largely cancelled out.

What is the optimal and/or maximum distance for a Tb-fluorescein pair?

The Förster radius, the distance at which energy transfer efficiency is half-maximal, is around 50-angstroms for the terbiumÆ fluorescein pair. However, the Förster radius does not give a complete indication of energy transfer efficiency when using long lifetime fluorophores such as terbium chelates. When using terbium chelates, energy transfer efficiency is determined by the distance of closest approach between the donor and acceptor during the excited state lifetime of the donor. In many assay systems, such as those designed using antibodies or peptides, there is a large degree of conformational freedom that allows the donor and acceptor to approach one another, effectively enhancing the FRET signal. Additionally, it is important to note that as the donor/acceptor pair approach one another and the efficiency of energy transfer increases, the fluorescent lifetime decreases to a comparable extent. From a practical standpoint, this means that when energy transfer is extremely efficient, FRET cannot be measured in time-resolved mode (because the energy transfer is complete before the measurement is made). This is another reason why TR-FRET assays based around terbium-labeled antibodies or streptavidin perform so well, because there exist a range of donor/acceptor distances, several of which are optimal for measuring FRET.

How many LanthaScreen assays can I run with a given amount of substrate?

It varies, depending on the concentration of substrate used in the assay. But in general, for the peptide substrates, 1 mg of peptide will run approximately 250,000 wells (10 µL reaction, 200 nM peptide). For Poly GT or GAT, the 1 mL of 30 µM size we sell is approximately 1 mg. With these substrates, 1 mL of 30 µM will run approximately 16,700 wells (10 µL reaction, 200 µM substrate).

20 nmol of our physiological protein substrates is sufficient for approximately 10,000 wells (10 µL reaction, 200 µM substrate).

I already have a biotinylated substrate that I know works with my kinase. Can I use this in a LanthaScreen assay?

Yes, a biotinylated substrate can be used in a LanthaScreen assay provided you have fluorescein-labeled streptavidin (available from Thermo Fisher Scientific Cat. No. SA100-02). A typical assay should contain 200-400 nM biotinylated substrate in the kinase reaction, and 1-2 nM Tb-labeled antibody and 50 nM fluorescein labeled streptavidin to detect the product. These conditions will need to be optimized for your particular system, but the recommended amounts are a good starting point. This format does, however, increase the cost associated with screening because of the additional component compared to the directly labeled substrate format. Also, it adds another layer of complexity with the additional component requiring optimization.

Should I use Poly-GT or Poly-GAT to assay my tyrosine kinase?

Try both or check our data to see if we have run your kinase of interest against these substrates (see reactivity chart at thermofisher.com/lanthascreen). In general, either substrate will work for a given tyrosine kinase. However, one of the substrates will probably show a lower EC50 with the kinase (i.e,. be a better substrate for the kinase), allowing less kinase to be used in the assay. Additionally, one substrate may show a larger assay window than the other (usually, but not always, Poly-GAT shows a larger window). However, either substrate will give an assay with an excellent Z' value.

Which substrate should I use to assay my tyrosine kinase?

If you want a specific peptide substrate, we offer two synthetic peptides determined to be effective substrates for src-family kinases; peptide 1 (YIYGSFK) and peptide 2 (KVEKIGEGTYGVVYK). Peptide 1 was a random peptide identified to be a pp60c-srcsubstrate and peptide 2 is a sequence from p34cdc2. Alternatively, fluorescein-labeled peptide substrates can be readily synthesized by us to suit your particular needs. In addition to peptide substrates, we offer fluorescein-labeled Poly-GT and Poly-GAT. We have had 100% success rate with these substrates to-date when assaying tyrosine kinases (TKs).

How many Lanthascreen assays can I run with a given amount of antibody?

This depends on the volume of the assay and the concentration of antibody used. At 2 nM antibody concentration in a final volume of 20 µL, 25 µg of antibody will run >4000 assays, and 1 mg of antibody will run >165,000 assays.

How much antibody should I use for my kinase assay?

In general, we recommend 2 nM as the final concentration of antibody in the detection reaction. The molecular weight of a Tb-labeled antibody is 150,000 Da so a concentration of a 2 nM solution corresponds to 0.3 µg/mL. This is a 1:3,300 dilution of a 1 mg/mL stock solution.

I would like to have my antibody directly labeled with the LanthaScreen terbium chelate. How do I do that?

There are a couple of different ways to directly label your antibody. First, you can have our custom labeling service prepare the material for you. You send us 1 mg of material or more and we will perform the labeling reaction for you. As an alternative to that, you may buy our terbium chelate in an amine or thiol reactive form and perform the labeling reaction in your lab.

I already have an antibody that I would like to use in a LanthaScreen assay. Is there a way I can determine if it will work without directly labeling it?

Yes, you may “indirectly” label your primary antibody by using a terbium- labeled, species-specific anti-IgG antibody. As a starting point for further optimization, we recommend a final concentration of 2 nM primary and 5 nM terbium-labeled secondary antibody in the assay well. The reaction conditions can be further optimized by increasing and/or decreasing the amount of terbium-lableled secondary antibody to determine the optimal amount to use. Check with the supplier of your primary antibody to determine its stock concentration. While in a majority of cases, the unlabeled primary and terbium-labeled secondary antibody may be pre-mixed prior to addition to the assay well, in some cases, separate additions (primary first, followed by secondary) are required. In general, assays using directly labeled antibodies may come to equilibrium faster or have a more stable signal (longer read window) than assays that use indirect labeling techniques. Also, although the response window (Z') is often acceptable when using an indirect labeling strategy, it may sometimes be improved by using a directly labeled primary antibody.

Which LanthaScreen antibody should I use?

For tyrosine kinase assays, we offer four different terbium-labeled anti-phosphotyrosine antibodies. When using fluorescein-labeled poly-GAT or poly-GT, performance using the Tb-labeled PY20 antibody is excellent. When using specific peptide substrates, performance may vary between the different varieties of anti phosphotyrosine antibodies, and as such we recommend evaluating the assay with a selection of antibodies in order to find the antibody that gives optimal performance. However, for tyrosine kinase assays, any of the anti-phosphotryrosine antibodies will likely perform well.

For serine/threonine kinases, we provide a reactivity chart at thermofisher.com/lanthascreen which shows the performance of various kinases using suitable substrate/antibody pairs.

How much substrate should I use in a LanthaScreen assay?

In general, 200 nM to 1 µM peptide or physiological protein substrate is often used in a LanthaScreen kinase assay. We recommend starting with 200 or 400 nM in the assay reaction. The molar concentration of substrate can be calculated from the molecular weight of the substrate, by dividing the mg/ml concentration by the molecular weight. This will give a molar concentration of the substrate. Multiplying by 1,000,000 will give a micromolar concentration. The molecular weight of each substrate can be found on the certificate of analysis for each individual substrate or on our website.

[Substrate] in µM = mg/mL/(molecular weight x 1,000,000)

How do I determine how much ATP to use in a LanthaScreen assay?

The amount of ATP to use depends on the goals of the specific assay. In an assay designed to identify ATP-competitive inhibitors, the assay is typically run at the ATP Km apparent. To determine the ATP Km apparent, perform the assay using a serial dilution of ATP and a constant amount of kinase and substrate. The EC50 value of this curve is the ATP Km apparent. Now, using the determined concentration of ATP and the same amount of substrate, run the assay using a dilution series of kinase. From the kinase titration curve, determine the EC80 concentration of kinase and use this concentration of kinase in subsequent assays.

When performing a kinase titration, why does the FRET ratio sometimes decrease at high concentrations of kinase?

At high concentrations of kinase, a decrease in signal ratio may be observed especially with tyrosine kinases. Because the kinase itself is often phosphorylated (or can be autophosphorylated), at high concentrations, it can be bound by the Tb-labeled antibody, thereby competing with fluorescein-labeled phosphorylated substrate for binding to the antibody. If this phenomenon is observed, it indicates that a lower concentration of kinase should be used in the assay.

How do I determine how much kinase to use in a LanthaScreen assay?

To determine the proper amount of kinase to use under a given set of substrate and/or ATP concentrations, you should perform the assay against a serial dilution of the kinase using a constant concentration of substrate and ATP. From the data generated, determine the EC80 (or the amount of kinase that will generate 80% of the maximal change in the FRET ratio). Use this amount of kinase for any additional optimizations. You may want to begin your kinase titration at a concentration lower than is typical in other kinase assay formats, as the LanthaScreen assay is extremely sensitive.

How does LanthaScreen perform in a screening environment?

Typical Z' values observed are 0.7 or greater.

Can I use LanthaScreen reagents in cell lysate assays, and do you have data or protocols for this application?

We have not yet developed products specific to cell-lysate based applications. However, we have shown in several cases that the Tb-based LanthaScreen signal is indeed stable in the presence of cell lysates. So, for customers building their own assays using the LanthaScreen Toolbox, the presence of cell lysate components is not expected to interfere with the LanthaScreen signal.

What applications are the LanthaScreen reagents appropriate for?

Terbium-labeled phosphospecific antibodies have been applied to assays for over 150 kinases. Antibodies specific to epitope tags such as 6-His or GST have been applied to nuclear receptor assays. Terbium labeled streptavidin is optimal for developing protease assays using biotinylated fluorescein labeled peptides. Ubiquitin assays for E3s have been developed using labeled ubiquitin proteins and anti-epitope tags, and deubiquitination assays can be performed using our DUB substrate. See the website (thermofisher.com/lanthascreen) for data and examples.

Are there specific plates I should use for my LanthaScreen assay?

The LanthaScreen biochemical assay format is suitable for nearly any brand or volume of assay plate. We have developed many assays using Corning low volume black polystyrene 384 well plates (#3676). We have also successfully used Corning low volume white polystyrene 384 well plates (Cat. No. 3673), Thermo LabSystems Microfluor 1 Black 96 well plates (Cat. No. 7005), and Thermo Electron Cliniplate black polypropylene 384 well Plates (Cat. No. 95040020). When performing the assay on monochromator-based readers such as the Tecan Safire2 or the Molecular Devices M5, assay performance may be increased by using white microtiter plates. With filter-based instruments, the differences seen between black and white plates are often negligible.

We recommend the use of white assay plates for LanthaScreen GFP Cellular Assays to achieve optimal signal detection. We have successfully used Corning low-volume white polystyrene plates (Corning Cat. No. 3674) and Corning tissue culture-treated white polystyrene plates (Corning Cat. No. 3704). The tissue culture-treated plate is recommended for assay formats that perform cell treatment, lysis, and detection in the same well/plate. The low-volume plate provides excellent results in assay formats where the cell lysate and terbium-labeled detection antibody are combined in a plate different from the cell cultivation plate. We expect LanthaScreen GFP Cellular Assays to be compatible with equivalent plates from all major manufacturers.

Certain LanthaScreen GFP Cell Lines, which exhibit poor adherence to standard tissue culture-treated plates, may require the use of poly L-lysine coated plates to prevent loss of cells during medium removal.

Can I use a monochromator-based instrument to read LanthaScreen assays?

We have found two monochromator-based instruments that work well with LanthaScreen biochemical assays; the Tecan Safire2 and the Molecular Devices SpectraMax M5. For the Safire2, we recommend exciting the samples at 332 nm with a 20 nm bandwidth. The donor (terbium) signal is measured at 485 nm with a 20 nm bandwidth and the acceptor (fluorescein) signal is measured at 515 nm with a 20 nm bandwidth. With the SpectraMax M5, we recommend exciting at 332 nm and measuring intensity at 488 and 518 nm (the band width is built in at 15 nm and cannot be adjusted).

When using a monochromator-based instrument, white assay plates often show better performance. In filter-based instruments, the color of the plate usually shows a negligible effect on data quality.

If you are using a LanthaScreen GFP Cellular Assay, we do not recommend the use of monochromator-based instruments, as the sensitivity of these instruments is not sufficient to adequately detect the endogenously expressed GFP fusion proteins.

Can I use a standard fluoresein filter to measure LanthaScreen emission?

In general, this is not possible. The typical fluorescein emission filter has a bandwidth that is too large for optimal performance in a LanthaScreen assay. Such filters not only measure the intensity of the acceptor fluorophore, but also signals from terbium itself. Having this “contaminating” intensity present in the data will make the acceptor signal only a small portion of the raw intensity. This will have a negative impact on the data.

What filters/dichroics do I need to read a LanthaScreen assay?

We recommend using a 340 nm excitation filter with a 30 nm bandwidth, but an excitation filter with similar specifications will perform well. The exact specifications of the emission filters are more important. In a LanthaScreen assay, the donor (terbium) intensity is measured using a 495 nm filter (10 nm bandwidth) and the fluorescein (acceptor) intensity is measured using a 520 nm filter (25 nm bandwidth). Filters suitable for performing LanthaScreen assays can be obtained from Chroma as filter set PV001, or from other vendors. A filter module for the BMG PHERAstar can be obtained direct from BMG Instruments.

The dichroic mirror should ideally have a cutoff of less than 490 nm. A dichroic mirror meeting this specification is available from Chroma as part of filter set PV001, or from the instrument manufacturer. In some instruments (Tecan instruments, for example), the fluorescein dichroic performs well but must be manually selected. In other instruments, a 50:50 mirror may be selected, but in general will not give as high performance as a dedicated dichroic. The LanthaScreen filter module from BMG contains an optimal dichroic mirror.

For Research Use Only. Not for use in diagnostic procedures.