Support

F.A.Q.

Protein Kinases

Profiling Services

Assay Kits & Development

Crystallography

Detection of Protein : Protein Interactions

Others

NanoBRET™ TE Intracellular Kinase Cell-Based Assay Services

Protein Kinases

QPack size of protein kinase products?
A

Starting from 5ug, our kinase products are available in 100, 200, 500, and 1000ug per vial. Some kinases are available in only 5ug. 50ug is minimum for inactive & inactive-mutant kinases. For a bulk amount, please inquire.

QI am interested in protein kinase which is not on the Carna's list.
A

Please inquire us. Some may be under development. Also, we can produce mutated forms of specific protein kinase upon request.

QWhat is the origin of Carna's kinase product?
A

All protein kinases among the list are human recombinant protein.

QHow are your protein kinases manufactured?
A

The majority of our kinases are manufactured using a baculovirus expression system, and a few use an E.coli. expression system. For an overview of our methods, please follow our kinase products page and click on the product name of interest to view the complete “Product description.”

QAre your kinases sold in an activated form?
A

Yes. If a kinase demonstrates low activity post expression or a lag phase after initiation of the kinase reaction, we activate it by ATP treatment, co-expression or incubation with its upstream kinase(s), GST-tag removal by protease, or another activation method, depending on the nature of the kinase. Please refer to our technical note, “Carna Coffee Break No.1” for details.

QCan I purchase inactive kinase products?
A

Yes. We sell inactive kinases and inactive mutant kinases related to the MAP kinase cascade. These can be used as substrates and in other applications. Please see the product list on our kinase products page.

QIs it possible to cleave the affinity tag from your kinases?
A

Yes. All of our kinases contain a cleavage site between the kinase sequence and the tag. The GST-tag can be cleaved using Precission protease (Cytiva), and the his-tag can be cleaved using TEV protease. Our biotinylated kinases contain the DYKDDDDK tag between the kinase and the biotinylated site, where the biotinylated site can be cleaved using enterokinase.

QHow many biotins do your biotinylated kinases contain?
A

All of the biotinylated kinases we sell contain a single biotin per molecule.

QHow do you assure the quality of your kinases?
A

We determine the purity of our kinases using SDS-PAGE gels; the activity is determined in a kinase assay by examining phosphorylation of its substrate.

QI’m concerned kinase activity may vary batch-to-batch.
A

Since we use a highly reproducible manufacturing method, and we monitor the activity of each batch, activity differences between batches is minor for most of our kinase products. If you would like to confirm any possible activity or purity difference between batches, please contact us.

QHow do you confirm the identity of your kinase products?
A

We perform mass spectrometry for peptide fingerprinting to confirm the correct sequence.

QHow can I obtain the sequence(s) of your kinase(s)?
A

By clicking on the specific kinase name on our product information page, you can view sequence information under the “AMINO ACID SEQUENCE” , shown at the top of the page.

QWhich types of kinase products are best suited for use in binding assays?
A

We recommend our biotinylated kinases for binding studies because of their high purity and small molecular weight label.

QDo your kinase products have associated Safety Data Sheets?
A

Our kinases do not require Safety Data Sheets, however most products are subject to the Cartagena Law. Please contact us if you would like to confirm whether the product you are interested in is subject to the Cartagena Law.

QWhere are your kinases manufactured?
A

All our kinases are manufactured at Carna Biosciences’ in-house laboratory. We do not outsource.

QHow are your kinases shipped?
A

All of our kinases are shipped on dry ice. As soon as you receive the shipment, please transfer the kinase(s) to a -80℃ freezer (or colder). Proper storage is essential to shelf life. Once you thaw the kinase, aliquot the remainder to avoid freeze thaw cycles.

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Profiling Services

QWhy are assays performed at an ATP concentration equal to the Km value?
A

For ATP-competitive inhibitors, the dependencies between the half maximal inhibitory concentration (IC50) and ATP concentrations are described by the Cheng-Prusoff equation (IC50 = Ki + Ki/Km×[ATP]). By representing the ATP concentration with its Km value, the IC50 reflects 2×the Ki value. Thus, IC50 values become a direct and comparable measure of affinity between the inhibitor and the investigated kinases.

QHow much does it cost for the service?
A

Please inquire us with the number of sample, kinase, and concentration. All assays are performed in duplicate basically.

QHow do I set the compound test concentration?
A

We recommend that you first measure % inhibition at 1-10μM, and then choose compounds with strong inhibitory activity. For follow up IC50 determinations, you can reference the results of their % inhibition to set appropriate test concentrations.

QHow do I check on the details of profiling service?
A

Please download the latest kinase profiling book from our website. Detailed information about each kinase, assay condition, and more can be checked on the book.

QHow do I prepare test samples?
A

The required volume of samples is 500uL each if less than 100 target kinases, 1000uL each for over 100 kinases. Please prepare samples in 100% DMSO, 100x of the highest test concentration you selected. Please click here for more details.

QIs it possible for Carna to store the compounds that have been tested for use in follow up tests?
A

Yes. As a rule, we typically discard compounds 3 months after the date of submitting the result report, so we will store your compound during that time period. If you wish to extend the time we keep the compounds, please contact us.

QWhat assay format(s) does Carna use and why?
A

We typically utilize Mobility Shift Assay (MSA) as a first choice because it is easy to operate, highly reproducible, and can detect both phosphorylated and non-phosphorylated substrates simultaneously. However, the substrates that can be used in MSA have only one phosphorylation site in the sequence and are limited to peptides with a pI near neutrality. If no such substrate is found, we choose a different detection method. Although there are exceptions, as a rule, ADP-Glo™ is chosen if the substrates are lipids and IMAP is chosen if the substrates are peptides including two or more phosphorylation sites. The majority of our profiling assays are performed using the MSA format.

QWhat is the utility of profiling at 1mM ATP?
A

The 1mM ATP profiling assays are useful in predicting the potential intracellular selectivity of your inhibitor using conditions approaching physiological ATP concentrations. Profiling at 1mM ATP can also assist to identify possible non ATP-competitive inhibitors when compared with IC50 data generated at ATP Km.

QHow many kinases are selectable for profiling?
A

342 kinases are profiling available. You can select from just one kinase.

QHow do I appy for the profiling service?
A

After an agreement is concluded, please fill out the application form and submit it to us via email.

QWhen will I receive my profiling result?
A

Normally, 2-3 weeks after the receipt of compounds, you will receive a result in an encrypted file via email.

QWhat does the "preincubation" profiling include?
A

The service incorporates a 30 minute pre-incubation at room temperature with the test compounds and the investigated kinase prior to measuring the activity in our standard method of MSA. This is useful to assess potencies of compounds with slow binding kinetics.

QIs the panel assay available?
A

Carna is offering four unique pre-selected panels. You can also select additonal kinases to the combination of a panel.

QWhat does “Km bin” mean, with reference to the ATP concentration?
A

For assays run at ATP Km value, we create a series of groups, or ‘bins’ of ATP concentrations near the Km, but round numbers. For example, if a kinase has an ATP Km value of 47 μM, it will be grouped with other kinases with similar ATP Km values and performed at 50 μM ATP. This ensures that each kinase assay is performed at an ATP concentration very close to the Km value, and at the same time allows for efficiency of running a large number of kinase assays simultaneously.

QWhat reference compounds are used for Carna’s profiling assays?
A

We use staurosporine for most kinase assays, and in other cases we utilize inhibitors that are specific to a given kinase. Clicking the target kinase on our profiling services page, you can quickly see which reference compound is used in any kinase assay of interest.

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Assay Kits & Development

QCan I order IMAPTM beads?
A

IMAPTM beads can be sold upon request.

QHow do I refer to the protocol of assay kit in advance of ordering?
A

You can download and check the protocol of each platform on our website.

QCan I order the component separately?
A

After your purchase of a kit, each component of the kit can be ordered separately.

QWhen will I receive the product?
A

Our assay kit is custom-made. Normally, 2-3 weeks for the delivery.

QIs there anything I have to prepare before using assay kits?
A

Additional materials are required for IMAPTM, ELISA, and TR-FRET kits. Please refer to the first page of each protocol.

QWhat type of plate I have to prepare?
A

Please refer to each protocol for the name of manufacturer and catalogue number of plate which Carna recommends.

QComponent of assay kit?
A

The component varies by a platform. Please click here for more details.

QWhat is the minimum order size of assay kits?
A

100dp per set for ELISA and 400dp for other platforms.

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Crystallography

QCan I have a detailed sequence of crystal-grade protein kinase in advance of ordering?
A

A sequence is partially disclosable. Please inquire.

QPack size of crystal-grade protein kinase products?
A

Please feel free to inquire.

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Detection of Protein : Protein Interactions

QAre ProbeX’s luciferase assays applicable to the detection of PPIs?
A

Yes. These assays have been used to detect signals not only for GPCR studies but also for protein protein interactions inside a cell, such as FKBP-FRB binding. Whether our split luciferase generates a detectable signal when measuring protein protein interactions relies on various factors---protein structural stability, expression level inside a cell, proximity of the two luciferase fragments during the interaction, etc. If you wish to develop a PPI assay by the use of our luciferase technology, please contact us.

QWhat should I use as the luciferase substrate?
A

We recommend using Carna’s Split Glow Cell Assay Reagent (PXR-SG001), developed exclusively for our Split Luciferase Assay. Its composition is optimized for this assay to generate the brightest luminescence signal. When detecting a signal inside a living cell (as opposed to a cell lysate), use D-Luciferin (approximately 1.0 mM) dissolved in PBS or HBSS reagent.

QWhat instrument should I use for reading?
A

Use any plate reader applicable to bioluminescence detection, or a tube-type luminometer. A detector with higher sensitivity is better.

QWhat are the advantages of our luciferase technology compared to other similar systems?
A

Emerald luciferase in our split luciferase assay shows higher stability inside a cell and can emit a signal fifteen times brighter than firefly luciferase. Using our Split Glow Cell Assay Reagent (PXR-SG001), which can reduce background emission, you can detect stimuli-responsive PPI signals with high sensitivity. Its ability to detect a signal in cells for GPCR screening in just ten minutes after ligand stimulation also indicates that ProbeX’s luciferase is highly useful for high throughput screening of compounds. Its applicability for imaging in living cells and animals additionally makes it a valuable tool in evaluating compounds in a living cell or animal.

QAre Carna’s luciferase assays applicable for imaging?
A

Yes. The emerald luciferase used in our split luciferase system emits a very strong signal, which enables you to image real time signals at a single cell level for several hours. Please see the video of GPCR-arrestin imaging data on our website. Use diluted D-Luciferin in medium (approximately 1.0 mM) for the substrate in a living cell. Please contact us for detailed assay conditions.

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Others

QPayment term?
A

The condition is T/T 30 days.

QHow can I place an order?
A

We have a 100% wholly-owned subsidiary in the US and also distributors in Europe and Asia. Please contact us for more details.

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NanoBRET™ TE Intracellular Kinase Cell-Based Assay Services

QCan a customer designate which tracer is used in the assay?
A

We validate our testing using the tracer Promega recommends for each specific kinase. We can attempt different tracers upon request as custom studies, however we can’t guarantee success using other tracers.

QHow can I find out which tracer is used in my assay?
A

As a general rule, we use the tracer recommended by Promega. We occasionally choose a different tracer if our in-house assay results indicate this is preferable. The tracer used will be indicated in the study report. If you have any tracer questions or are interested in a kinase assay not listed on the Promega website, please contact us.

QWhat is the concentration of the tracer used in Residence Time determinations?
A

As a general rule, the tracer concentration is set at 2µmol/L in cases where the tracer concentration used in the IC50 determination exceeds 1µmol/L. Otherwise, it is set at 1µmol/L.

QIs it possible to disclose the name of reference compound in advance?
A

Yes, please inquire.

QDo you accept assay requests for kinases not listed in the current portfolio?
A

Yes, it may be possible. Please contact us.

QWhere can I find information regarding the position of NanoLuc® in the fusion kinases?
A

NanoLuc® may be fused to either the N-terminus or C-terminus of the kinase. Referring to the column titled Vector Name, accessed on Promega website, kinases fused to NanoLuc® on N-terminus are indicated as “NanoLuc®-kinase name”, kinases fused to NanoLuc® at the C-terminus are indicated as “kinase name-NanoLuc®”. The name of the vector will be indicated in the study report. Please contact us if you are interested in any kinase(s) not listed on the Promega website.

QWhy is a compound concentration of IC80-90 recommended for Residence Time determinations?
A

Ideally, the dissociation of bound compound from the kinase should be evaluated when compound occupies 100% of the kinase with no residual unbound compound. However, if the compound concentration is set beyond IC80-90, it might become difficult to remove the residual unbound compound completely by wash, which could affect accurate measurement. Therefore, it is recommended to perform the testing at the IC80-90 concentration.

QIs it possible to see an example of the report and data?
A

Yes, sample reports are available. Please inquire.

QWhat is the concentration of tracer used for IC50 determinations or % inhibition studies?
A

In general, we follow the application notes shown on Promega’s website above. Typically, the concentration is around the EC50 of the tracer dose response curve. The upper limit is 2 µmol/L.

QHow is the BRET ratio calculated?
A

The BRET ratio is calculated following the equation below using the donor signal (NanoLuc® fused kinase) and the acceptor signal (tracer).

    BRET ratio (mBRET)

    = {(Acceptor_sample/Donor_sample) – (Acceptor_no tracer/Donor_no tracer)} ×1000

    Acceptor_sample: The acceptor signal of the sample or DMSO control in the presence of tracer.
    Donor_sample: The donor signal of the sample or DMSO control in the presence of tracer.
    Acceptor_no tracer: The acceptor signal of DMSO control in the absence of tracer.
    Donor_no tracer: The donor signal of the DMSO control in the absence of tracer.

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Please refer for the other questions from here.

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