Thermo Fisher Scientific Safire2- Omnia Installation guide

Brand
Thermo Fisher Scientific
Model
Safire2- Omnia
Type
Installation guide
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 1 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
Omnia® Assay Setup Guide on the
Tecan Safire2™ Microplate Reader
NOTE: The Tecan Safire2™ 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. 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 Tecan instruments or software, please contact Tecan at 1-888-798-
0538 or [email protected].
A. Recommended Optics
wavelength (nm) diameter (mm)
Excitation 360/12 monochromator
Emission 1 485/12 monochromator
B. Instrument Setup
1. Make certain plate reader is turned on, and open up XFluor Data Manager
software on computer.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 2 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
2. When XFluor opens, select "Edit Measurement Parameter" from the drop-down
menu bar.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 3 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
3. A Measurement Parameter window will appear. Select "Fluorescence Intensity"
and "Kinetic".
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 4 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
4. Now select the “Plate” tab at the top of the window. When the new tab opens,
click on the "Browse" tab. Note this is also where to select which wells to read if
only reading part of the plate.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 5 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
5. When the new plate window appears, select your plate as shown below. Click
OK when finished to return to the main protocol setup window.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 6 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
6. Select the "Wavelengths" tab from the top. Next, click the "Edit Wavelength
Table" button.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 7 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
7. A new popup will appear. If you do not have suitable settings already loaded,
enter your excitation and donor emission wavelengths in the blanks below
"Name", "Ex", and "Em", respectively. When you have entered the desired
settings, select "Insert" to enter then into the table of defined settings as shown,
and then select OK when finished.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 8 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
8. When returned to the main protocol window, set your excitation and emission
bandwidths as shown below, then select the "Meas. Params" tab.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 9 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
9. Set the Gain for “Optimal”, 10 reads, and set the Read Mode to "Bottom". When
finished, select OK.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 10 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
10. You will be returned to the Multi-Labeling window. Check the "Do Measurement
2" box and click the "Edit Measurement Parameter" tab to enter your excitation
and acceptor settings.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 11 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
11. Both the "General" and "Plate" tabs will now be locked, as their settings were
already determined by the first set of measurement parameters entered. Go
straight to the "Wavelengths" tab, and select "Edit Wavelength Table" as before.
In the popup, enter the values for your excitation and your ac ceptor emission as
shown below, and again select "Insert" to enter them into your tables, then select
OK.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 12 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
12. You will return to the main protocol page again. Enter the appropriate instrument
bandwidths, then select the "Meas. Params" tab.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 13 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
13. Make sure the Gain is set to "Optimal" and the Read Mode is set to "Bottom".
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 14 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
14. At this point you will default all the way back to the Multi-Labeling window. If you
have not already, insert your plate, and select the "Run" tab to read.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 15 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
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
123456789101112
JH1/JH2 AStaurosporine JAK2 In II No inhib. no kinase
B100% Phos
V617F CStaurosporine JAK2 In II No inhib. no kinase
D
E
F
G
H
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 16 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
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.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 17 of 17
Setup Guide on the Tecan Safire2™ 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: drugdiscoverytech@invitrogen.com
D. Results:
Figure 2: Omnia® Kinase Assay. Omnia® assay performed as described above and
read on the Tecan Safire2™. 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 + 235.46
200
250
300
350
400
450
500
0 102030405060
Staurosporine
JAK 2 Inhi bi tor II
Kinase
Linear (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 + 2165.8
y = 0.0843x + 2848
2000
2500
3000
3500
4000
4500
5000
0 102030405060
JAK2
JH1/JH2
y = 3.8393x + 274.78
y = 0.5793x + 234.81
y = 0.1208x + 235.46
200
250
300
350
400
450
500
0 102030405060
Staurosporine
JAK 2 Inhi bi tor II
Kinase
Linear (Kinase)
Linear (JAK2 Inhibitor II)
Linear (Staurosporine)
JAK2
JH1/JH2
y = 3.8393x + 274.78
y = 0.5793x + 234.81
y = 0.1208x + 235.46
200
250
300
350
400
450
500
0 102030405060
Staurosporine
JAK 2 Inhi bi tor II
Kinase
Linear (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 + 2165.8
y = 0.0843x + 2848
2000
2500
3000
3500
4000
4500
5000
0 102030405060
98.90.650JAK2 Inhibitor II
99.80.107Staurosporine
057.766Kinase
% InhibitionRate (RFU/min)Rxn Cond.
98.90.650JAK2 Inhibitor II
99.80.107Staurosporine
057.766Kinase
% InhibitionRate (RFU/min)Rxn Cond.
JAK2
JH1/JH2
V617F
y = 3.8393x + 274.78
y = 0.5793x + 234.81
y = 0.1208x + 235.46
200
250
300
350
400
450
500
0 102030405060
Staurosporine
JAK 2 Inhi bi tor II
Kinase
Linear (Kinase)
Linear (JAK2 Inhibitor II)
Linear (Staurosporine)
y = 29.588x + 2164.2
y = 2.821x + 2240.1
y = -1.1374x + 2902.7
2000
2500
3000
3500
4000
4500
5000
0 102030405060
JAK2
JH1/JH2
V617F
y = 3.8393x + 274.78
y = 0.5793x + 234.81
y = 0.1208x + 235.46
200
250
300
350
400
450
500
0 102030405060
Staurosporine
JAK 2 Inhi bi tor II
Kinase
Linear (Kinase)
Linear (JAK2 Inhibitor II)
Linear (Staurosporine)
JAK2
JH1/JH2
V617F
y = 3.8393x + 274.78
y = 0.5793x + 234.81
y = 0.1208x + 235.46
200
250
300
350
400
450
500
0 102030405060
Staurosporine
JAK 2 Inhi bi tor II
Kinase
Linear (Kinase)
Linear (JAK2 Inhibitor II)
Linear (Staurosporine)
y = 29.588x + 2164.2
y = 2.821x + 2240.1
y = -1.1374x + 2902.7
2000
2500
3000
3500
4000
4500
5000
0 102030405060
y = 57.766x + 1902.9
y = 0.6504x + 2097.9
y = 0.1065x + 2697.2
2000
2500
3000
3500
4000
4500
5000
0 102030405060
Staurosporine
JAK2 Inhibitor II
Kinase
Linear (Kinase)
Linear (JAK 2 Inhibitor II)
Linear (Staurosporine)
y = 40.637x + 2100.7
y = 3.6401x + 2304.9
y = -1.2864x + 2946.2
2000
2500
3000
3500
4000
4500
5000
0 102030405060
Staurosporine
JAK2 Inhibitor II
Kinase
Linear (Kinase)
Linear (JAK 2 Inhibitor II)
Linear (Staurosporine)
91.13.640JAK2 Inhibitor II
103.2-1.286Staurosporine
040.637Kinase
% InhibitionRate (RFU/min)Rxn Cond.
91.13.640JAK2 Inhibitor II
103.2-1.286Staurosporine
040.637Kinase
% InhibitionRate (RFU/min)Rxn Cond.
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Thermo Fisher Scientific Safire2- Omnia Installation guide

Brand
Thermo Fisher Scientific
Model
Safire2- Omnia
Type
Installation guide
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