Omega Omnia kl10Mar09 - Thermo Fisher ScientificOmnia® Assay Setup Guide on the BMG LABTECH Omega Microplate Reader NOTE: The BMG LABTECH Omega Microplate Reader was tested for compatibility

Omnia® Compatible Microplate Reader Documentation

Version No.: 10 Mar 09 Page 1 of 10

Setup Guide on the BMGLABTECH Omega 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

BMG LABTECH Omega Microplate Reader NOTE: The BMG LABTECH Omega 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 BMG LABTECH instruments or software, please contact BMG LABTECH at 1-877-264-5227 or www.bmglabtech.com. A. Recommended Optics

wavelength (nm)

BMG LABTECH Filters

Excitation

360 (or similar) *contact BMG LABTECH

Emission 1

485 (or similar) *contact BMG LABTECH

B. Instrument Setup

1. Make certain plate reader is turned on, and open up Omega Control software on computer. Insert plate into plate reader.






2. When Omega Control software opens, if you do not have a pre-existing protocol for Omnia®, select "Test Protocol" from the Test Setup menu bar at the top of the window.






3. At this point, a new screen will open (below). Click on the “Show all test protocols” button on the left side of the screen, then select “New” from the tabs at the bottom.






4. A new window will pop up. Select “Fluorescence Intensity” and "Plate mode (slow kinetics)"” and then select “OK”.






5. A new Protocol window will open automatically. Enter a test name, select plate type, and select the Optic type. Next, set the number of flashes per well and select the excitation and emission filter from the drop-down tabs. In the Kinetic window, enter 61 cycles, 10 flashes per well and cycle, and 60-second intervals to prepare a kinetic assay of 1 hour with readings taken every 60 seconds.






6. Select the "Layout" tab at the top of the window. Select the wells you wish to read by dragging over them. When finished, select "OK.






7. A new window will appear. Select the wells you wish to read by highlighting them. When finished, select "OK"






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. This section is only an explanation of the procedure followed in generating the data for Section D and not recommended as a control protocol. We recommend all first-time users follow the provided protocols and/or validation packets specific for their assay, and run proper controls. 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 and JAK2 Inhibitor II were 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

1 2 3 4 5 6 7 8 9 10 11 12JH1/JH2 A Staurosporine JAK2 In II No inhib. no kinase

B 100% PhosV617F C Staurosporine JAK2 In II No inhib. no kinase

DEFGH






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.

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:

Figure 2: Omnia® Kinase Assay. Omnia® assay performed as described above and read on the BMG LABTECH OPTIMA. 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)

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


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

y = 44.697x + 2174.7

y = 3.7849x + 2165.8y = 0.0843x + 2848

2000

2500

3000

3500

4000

4500

5000

0 10 20 30 40 50 60

90.8480.53JAK2 Inhibitor II

100.14-1.31Staurosporine

0878.81Kinase

% InhibitionRate (RFU/min)Rxn Cond.



0878.81Kinase


JAK2 JH1/JH2V617F

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


JAK2 JH1/JH2V617F

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


y = 29.588x + 2164.2

y = 2.821x + 2240.1y = -1.1374x + 2902.7

2000

2500

3000

3500

4000

4500

5000

0 10 20 30 40 50 60

y = 379.58x + 4968.1

y = -6.9763x + 5562.3

y = 13.784x + 4915.1

2000

7000

12000

17000

22000

27000

32000

0 10 20 30 40 50 60




0379.58Kinase




0379.58Kinase


y = 878.81x + 11022

y = 80.526x + 8001.2

y = -1.3121x + 8738.3

2000

7000

12000

17000

22000

27000

32000

0 10 20 30 40 50 60


Documents

Omega Omnia kl10Mar09 - Thermo Fisher ScientificOmnia® Assay Setup Guide on the BMG LABTECH Omega Microplate Reader NOTE: The BMG LABTECH Omega Microplate Reader was tested for compatibility