FLx800 Omnia kl10Mar09 - Thermo Fisher Scientific€¦ · C. Omnia® Kinase Assay using JAK2 JH1/JH2 and JAK2 JH1/JH2 V617F NOTE: The following is a sample titration assay performed

Omnia® Compatible Microplate Reader Documentation

Version No.: 10 Mar 09 Page 1 of 17

Setup Guide on the BioTek Instruments FLx800 Fluorescence™ Microplate Reader

Have a question? Contact our Technical Support Team

NA: 800-955-6288 or INTL: 760-603-7200 Select option 3, ext. 40266 Email: [email protected]

Omnia® Assay Setup Guide on the

BioTek Instruments FLx800™ Fluorescence Microplate Reader NOTE: The BioTek Instruments FLx800™ Fluorescence Microplate Reader was tested for compatibility with Invitrogen's Omnia® Assay using the Omnia® Y Peptide 12 kit (KPZ3121) and JAK2 JH1/JH2 and JAK2 JH1/JH2 V617F kinases. The following document is intended to demonstrate setup of this instrument and provide representative data. For more detailed information and technical support of Invitrogen assays please call 1-800-955-6288, select option "3", then extension 40266. For more detailed information and technical support of BioTek instruments or Gen5 software, please contact BioTek Instruments at 1-888-451-5171. A. Recommended Optics

BioTek Instruments part number

wavelength (nm)

diameter (mm)

Excitation (7082220)

360/40 (or similar) 18

Emission 1 (7082221)

485/20 (or similar) 18

B. Instrument Setup

1. Make certain plate reader is turned on, and open up BioTek Gen5 software on computer.






2. When Gen5 opens, if you do not have a pre-existing protocol for Omnia®, select "Protocol" in the "Create a New Item" menu near the center of the screen.






3. At this point, a blank protocol will open (below). To ensure that the correct instrument is selected, click on "Reader Configuration" under tools.






4. Check that the instrument selected is correct (below). If not, select "Delete" to remove the existing instrument, and then "Add".






5. Upon selecting "Add" a popup window will appear allowing you to select your instrument from the drop-down menu. Select your instrument here. Click on "Test Communication" to verify that the instrument is communicating properly with the software. When this is done, click on "Setup" finally to check filter and dichroic settings.






6. Select the "Fluorescence/Luminescence" tab at the top of the popup window. Enter the correct values for your filters present in your filter holder(s). Make certain to do so in the proper positions. Note if you have more than one filter holder for excitation or emission you may have to check that the correct set listed in the settings or enter the appropriate one. Click on "Send Values" to load the filter positions and settings into the instrument.

7. Select "Mirrors" and repeat for the dichroic mirror as well, if needed. Select

"Close" when finished.






8. Since you have already selected to open a new protocol, at this point the instrument and software are ready to set up a new protocol. Select "Procedure" on the left hand side and a new window will open up. Select plate type (NOTE for Omnia® assays, plates are supplied).






9. Select the "Read" tab. Select "Fluorescence" and "Endpoint" from the drop-down menus. Select the proper filters and dichroic mirror settings, as shown below. Also, select the wells of your plate you wish to read by clicking on the "Full Plate" icon to the left (A1-D12 already selected).






10. Since Omnia® assays are run in kinetic mode, you will still need to enter kinetic parameters. Click on the "Kinetic" tab within the "Procedure" box to open the following window. Select 1 hour for the run time, and 1 minute intervals. Click "OK".






11. Omnia® assays are also run at 30 degrees. Select the "Set Temperature" tab and set the reader incubator to 30ºC and click "OK".






12. Your screen should now look like the picture below. Select "Validate" to ensure all steps are correct, and then select "OK". In the upper left, under "File", save your protocol.






13. To run an actual experiment, click on "File" again, and select "New Experiment" from the drop-down list. A smaller window will appear; select your protocol and click "OK".






14. A new window will again appear; this is the experiment window. Insert your plate to be read and click on the "Read Plate" icon with the small green arrow (second from last icon in the lower toolbar).






15. One last popup will appear. Select "Read" and instrument will commence reading your plate. NOTE: There are several options for collecting data from the BioTek Gen5 software. Data can be exported directly to excel, or appropriate macros can be prepared within the BioTek software. For more information on data output options, please contact BioTek Technical Support.






C. Omnia® Kinase Assay using JAK2 JH1/JH2 and JAK2 JH1/JH2 V617F NOTE: The following is a sample titration assay performed for demonstration purposes. The instrument settings above would be sufficient for any Omnia® kinase assay, the information below is provided as representative data. Assay was run in 50 µl using 1 µM kinase inhibitor and at a kinase concentration of 435 ng kinase per well, based upon recommendations from R&D. Concentration of kinase used will vary, for more information please see the Omnia® kinase assay protocol for your specific kinase of interest at the following link: http://www.invitrogen.com/content.cfm?pageid=11338&cid=fl-OMNIA.

1. Prepare initial inhibitor solutions at 10X from stock, in deionized water. Add 5 µl

10 µM inhibitor to proper wells. Note in the plate outline shown below, 5 µl Staurosporine added to wells A1-A3 and C1-C3 and 5 µl JAK2 Inhibitor II added to wells A4-A6 and C4-C6. Add 5 µl water alone to wells B1-B6, B10-B12, and D1-D3 (see Figure 1).

Figure 1: Schematic of Omnia® plate layout. Staurosporine, JAK2 Inhibitor II, JAK3 Inhibitor, and AG-490 were all assayed at 1 µM in triplicate against both JAK2 JH1/JH2 and JAK2 JH1/JH2 V617F. Note no inhibitor controls were also run for both kinases to determine full kinase activity, as well as a kinase-free control and a phosphopeptide control.

2. Master Mix minus inhibitor prepared as follows (quantities are per well, in this

case multiplied by 26 to allow for 24 reactions plus excess): Kinase Reaction Buffer (10X) 5 µl Tyrosine Peptide Substr. (dilute to 10X) 5 µl ATP Solution (dilute to 10X) 5 µl DTT Solution (dilute to 10X) 5 µl Deionized Water 20 µl

3. Add 40 µl Master Mix per well to wells A1-A6, B1-B6, C1-6, and D1-D3. Add 5 µl Omnia® Tyr Phosphopeptide Control and 5 µl all other components above except substrate to wells B10-B12, and deionized water to a total of 50 µl.

1 2 3 4 5 6 7 8 9 10 11 12JH1/JH2 A Staur. JAK2 Inh. II

B No Inh. No Kinase 100% Phosp.JH1/JH2 C Staur. JAK2 Inh. II

V617F D No Inh.EFGH






4. Allow plate to pre-incubate 5 minutes at 30º C.

5. Add 5 μL JAK2 JH1/JH2 to wells A1-A12 and B1-B3, and 5 µl JAK2 JH1/JH2

V617F to wells C1-C12 and D1-D3 (both kinases pre-diluted to 85 µg/ml).

6. Read and analyze as directed in the protocol.






D. Results:

Rxn Cond. Rate (RFU/min) % Inhibition

Kinase 3.5744 0 Staurosporine 0.0782 96.8

JAK2 Inhibitor II 0.9732 74.1

Rxn Cond. Rate (RFU/min) % Inhibition

Kinase 3.839 0 Staurosporine 0.121 96.8

JAK2 Inhibitor II 0.579 79.4

Figure 2: Omnia® Kinase Assay. Omnia® assay performed as described above and read on BioTek Instruments FLx800™. Assay results monitored at 1-minute intervals, data graphed in Microsoft Excel.

JAK2 JH1/JH2

y = 3.8393x + 274.78

y = 0.5793x + 234.81

y = 0.1208x + 235.46200

250

300

350

400

450

500

0 10 20 30 40 50 60

StaurosporineJAK2 Inhibitor IIKinaseLinear (Kinase)Linear (JAK2 Inhibitor II)Linear (Staurosporine)

y = 3.5744x + 295.7

y = 0.9732x + 219.74

y = 0.0782x + 233.13

y = 3.5744x + 295.7

y = 0.9732x + 219.74

y = 0.0782x + 233.13

JAK2 JH1/JH2 V617F

y = 3.8393x + 274.78

y = 0.5793x + 234.81

y = 0.1208x + 235.46200

250

300

350

400

450

500

0 10 20 30 40 50 60

StaurosporineJAK2 Inhibitor IIKinaseLinear (Kinase)Linear (JAK2 Inhibitor II)Linear (Staurosporine)

Documents

FLx800 Omnia kl10Mar09 - Thermo Fisher Scientific€¦ · C. Omnia® Kinase Assay using JAK2 JH1/JH2 and JAK2 JH1/JH2 V617F NOTE: The following is a sample titration assay performed