Plate Readers

The LanthaScreen® TR-FRET assays can be performed on a variety of plate readers, including those from Tecan (ULTRA, Infinite® F-500, Infinite® M1000, and Safire2™), Molecular Devices (Analyst®), BMG LABTECH (PHERAstar Plus), and PerkinElmer (EnVision®, Victor™, and ViewLux™) or any other plate reader configured for LANCE® or HTRF® assays. Monochromator-based instruments such as the SpectraMax® M5 Multi-Mode  Microplate Reader or Safire2™ may not be suitable for some of the LanthaScreen® Kinase Binding Assays with smaller assay windows.


Proper instrument setup is crucial for successful LanthaScreen® assays. For instrument-specific setup guides, please see our step-by-step setup documents. Recommendations differ slightly among instruments; check the details carefully, particularly with regard to filter bandpass and recommended dichroic mirrors.

Settings common to all Eu or Tb LanthaScreen® assays

Note:
These settings differ from those for other commercially available TR-FRET assays and are optimized for LanthaScreen® assays.

Excitation 340 nm (30 nm bandwidth)
Delay time 100 μs
Integration time 200 μs


For additional assistance or more information on performing these assays, contact your Invitrogen representative or Invitrogen Discovery Sciences Technical Support at +1 760 602 6500 (select option 3 and enter 40266), or email drugdiscoverytech@lifetech.com.

Plates

Assays are typically performed in black, low-volume 384-well plates (Corning Part # 3676). However, white plates may yield higher-quality data (Corning Part # 3673) for assays that have a relatively low assay window. White plates are essential for assays that are being measured on monochromator-based instruments or older filter-based instruments.

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Molecular weight information and conversions

Our calculations use the equation V1C1 = V2C2, where V denotes volume, C denotes concentration, and the numbers 1 and 2 denote initial and final conditions, respectively.

An important conversion: The number of μg equal to the molecular weight expressed in kDa is equal to 1 nmol.

Examples:


Molecular weight in kDa Conversion
LanthaScreen® antibody 150 kDa 1 nmol = 150 μg
Src 62.3 kDa 1 nmol = 62.3 μg
Abl2 130.9 kDa 1 nmol = 130.9 μg
Fluorescein-CREBtide substrate 2.076 kDa 1 nmol = 2.076 μg



Kinases

Epitope tags such as GST and His are always considered when calculating the molecular weight of a kinase. For any kinase complexes (e.g., cyclin/CDK dimer), the molecular weight of the complex should be used. For example, the molecular weights of CDK9 and cyclin K are 46.9 kDa and 44.9 kDa, respectively, for a sum of 91.8 kDa. The 91.8 kDa value is used to convert mg/mL to nM.

The V1C1 = V2C2 equation describes how the amount of material in a solution is the same before and after a dilution is performed. The amount can be represented as a mass (μg, ng, etc.) or in moles (μmol, nmol, etc.).

Kinases from Invitrogen are typically supplied at 0.1 to 0.5 mg/mL. To determine how much kinase to use in the binding assay, you must first convert the concentration in mg/mL to nM. The molecular weight is listed on the kinase Certificate of Analysis (COA).


Note:  If you are not familiar with this equation, learn more by entering “V1C1 = V2C2” as the query in your favorite search engine.

Note: COAs are also available on the product purchase page. Enter the part number or kinase name in the red search box at the top of any Invitrogen web page. Select the specifications tab. Click on Certificates of Analysis and then click search (without entering a lot number).


Example: Src kinase

Src has a molecular weight of 62.3 kDa. Therefore, 62.3 μg = 1 nmol.

If the concentration listed on the tube for lot 26726QQ is 0.58 mg/mL, the conversion is:

0.58 mg    x     1,000 μg     x     1 nmol       x      1,000 mL      =    9,300 nM     
    mL                     mg               62.3 μg                      L   


Using V1C1 = V2C2, calculate the volume of kinase needed to prepare the dilution.

For example, to prepare 1,000 μL of 15 nM Src kinase solution:

V1 x 9,300 nM = 1,000 μL x 15 nM; therefore, V1 = 1.6 μL


Because it is difficult to accurately pipet less than 2.0 μL of any kinase solution prepared in glycerol, we recommend preparing an intermediate 1:10 dilution using 1X kinase reaction buffer. Remember to recalculate the volume required for the diluted concentration of the kinase.

For example, dilute 2 μL of kinase with 18 μL of 1X Kinase Buffer A.

Perform the calculations again using a 1:10 dilution of kinase, so 930 nM instead of 9,300 nM:

V1 x 930 nM = 1,000 μL x 15 nM; therefore, V1 = 16.1 μL

Take 16.1 μL of diluted kinase to a final volume of 1,000 μL to prepare a 15 nM solution of kinase (which includes antibody for the binding assay).

Keep all diluted kinases on ice.

Antibody

Note:
Antibodies to GST or His tags are generically referred to as anti–epitope tag antibodies.

All LanthaScreen® antibodies are 150 kDa. The Tb or Eu chelate adds an insignificant mass, so it is not necessary to consider their molecular weight. Since all antibodies are 150 kDa, the following general guidelines apply. Check the tube to determine the antibody concentrations. Standard LanthaScreen® antibody concentrations are:

1.0 mg     =     6.7 μM   =   6,700 nM
  mL

0.5 mg     =     3.3 μM   =   3,300 nM
  mL

0.25         =      1.7 μM  =    1,700 nM
  mL


Peptide substrate

Many fluorescein-labeled peptides are supplied at 1 mg/mL and must be converted to nM to determine how much to use in an assay. This conversion requires the molecular weight, which is supplied in the Certificate of Analysis (COA). For example, Fl-CREBtide (PV3508) has a molecular weight of 2,076 Da.

2,076 Da = 2.076 kDa; therefore, 1 nmol = 2.076 μg

1 mg    x     1,000 μg     x     1 nmol       x      1,000 mL    x       1 μmol           =      9,300 nM     
  mL                 mg                2.076 μg                    L                   1,000 nmol


The concentration of the fluorescein-CREBtide substrate is 480 μM.

The 1X, 2X, 3X, and 4X notations

LanthaScreen® assays are addition-only assays; nothing is ever removed from an assay well. You add reagent 1, then reagent 2, etc. Each subsequent addition dilutes each other reagent. Remember to account for this dilution while preparing components.

For example:

 Volume addedFraction of total volumePrepared at
Reagent A2.5 μL1/44X
Reagent B5.0 μL1/22X
Reagent C2.5 μL1/44X

The term 1X refers to the final concentration of a component for a particular reaction; a reagent prepared at 4X strength refers to the extent of dilution (1:4) required to achieve the final concentration in the assay.

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Compound Serial Dilutions

Creating the 100X compound dilution series

A dilution series of test compound or control inhibitor in 100% DMSO is first prepared at 100 times the concentrations to be assayed. By performing the initial dilutions in 100% DMSO, you can minimize solubility problems associated with dilutions into aqueous buffer and stabilize the IC50 values.

For the control inhibitor and test compounds, we recommend 10- to 16-point curves with serial dilution steps ranging from a factor of 4 (4-fold) to a factor of 2 (2-fold). Fewer points or higher-fold dilutions are not recommended. Example of a 16-point 2-fold dilution series: A “master” dilution series of control inhibitor or test compound can be prepared in two separate 8-tube PCR strips (or in a 96-well plate), and stored at –20°C or –80°C for use in future experiments. The dilutions are “staggered” between strips, as shown on the left side of Figure 5. Two strips are used to facilitate the eventual transfer from a 96-well plate to a 384-well plate.

  1. Add 50 μL of DMSO to tubes 2–8 of strip A and all tubes of strip B.

  2. Add 100 μL of control inhibitor or test compound in DMSO at 100X the highest concentration to be tested to tube 1 of strip A.


  3. Example: If preparing staurosporine for use with the Src training assays, add 10 μL of 10 mM staurosporine to 90 μL DMSO in well A1 for a 100X concentration of 1 mM. Mix well by pipetting up and down.

  4. Transfer 50 μL of inhibitor from tube 1 of strip A to tube 1 of strip B.

  5. After mixing, transfer 50 μL from tube 1 of strip B to tube 2 of strip A.

  6. This process is repeated for all but the final tube of strip B, which contains only DMSO (no inhibitor). Discard 50 μL from the last well (A8) containing inhibitor, so that each well contains an equal volume.


Creating intermediate dilutions

Note:
Intermediate dilutions are required to keep the DMSO concentration constant at 1% across the dilution series.

An intermediate dilution from the 100X dilution series is necessary prior to performing a LanthaScreen® Kinase Assay. Test compounds or control inhibitors for the LanthaScreen® Activity Assay are diluted to 4X, and for the LanthaScreen® Eu Kinase Binding Assay diluted to 3X.

Intermediate dilutions of test compound or control inhibitor are prepared in 1X kinase buffer plus any additives for the LanthaScreen® Activity Assay. Kinase Buffer A diluted to 1X consists of 50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35.

To prepare these dilutions manually, we recommend volumes of no less than 4 μL for pipetting accuracy. See each assay section for more details.




Figure 5.
Preparing a 100X dilution series of test compounds or control inhibitor. A typical starting concentration in A1 is 1 mM. Dilutions are performed in 100% DMSO.

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LT129       1-Jan-2010