omnia assay setup guide on the - thermo fisher...

Omnia® Compatible Microplate Reader Documentation

Version No.: 10 Mar 09 of 13

Setup Guide on the BMGLABTECH PHERAstar/PHERAstarPlus Microplate Readers

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 PHERAstar/PHERAstarPlus Microplate Readers NOTE: The BMG LABTECH PHERAstar/PHERAstarPlus Microplate Readers were 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 Optic Module

Excitation

360 (or similar) *contact BMG LABTECH

Emission 1

485 (or similar) *contact BMG LABTECH

Dichroic Mirror *contact BMG LABTECH

B. Instrument Setup

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






2. When PHERAstar Control software opens, if you do not have a pre-existing protocol for OmniaTM, 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” or "Fluorescence Intensity" 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 “Endpoint” and then select “OK”.NEED PIC FOR PHERAstar here






5. A new Protocol window will open automatically. Enter a test name and select plate type. From the drop-down menu, select your optic module. Because Omnia is a kinetic assay, enter the desired number of cycles and the desired cycle duration. In this case we set up a 60-minute assay with one-minute read intervals. When finished, select the "Layout" tab at the top of the Protocol window.






6. Select the wells you wish to read. When finished, select OK.






7. You will return to the initial settings window. From the drop-down menus at the top, select "Measure" and "Measure" again.






8. A new window will appear allowing you to select which of your test protocols you wish to run. Select the protocol you created for Omnia, and then press OK.






9. A new window will appear. Place your plate in the reader, and select a well to use for adjusting gain and focus by highlighting the well of your choice. The gain or sensitivity can be adjusted at this point, in this case a positive control (phosphopeptide) should be used to avoid going off scale during the assay. When finished, click on the "Start Adjustment" tab.

10. In a moment, the instrument will have calculated it's optimal focal height and the gain adjustments necessary. When finished, click on the "Start Measurement" tab to read.






11. When PHERAstar is done reading, you can collect your data by clicking “Results” on the toolbar at the top of the window. This will automatically redirect you to a Microsoft Excel file which collects run data. Select your run of interest from the list to open, and then select the "Raw Data" tab at the bottom to view data in a plate layout format.






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 PHERAstar. 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

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

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


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

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

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 = 1070.1x + 14357

y = -21.626x + 17243

y = -6.0781x + 17387

2000

12000

22000

32000

42000

52000

62000

0 10 20 30 40 50 60


y = 8079.7x + 50716

y = -44.494x + 37321

y = -28.271x + 47540

2000

22000

42000

62000

82000

102000

122000

142000

0 10 20 30 40 50 60


102.3-21.63JAK2 Inhibitor II

100.6-6.08Staurosporine

01070.1Kinase

% InhibitionRate (RFU/min)Rxn Cond.



01070.1Kinase




08079.74Kinase




08079.74Kinase


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