Thermo Fisher Scientific VarioskanFlash- Omnia Installation guide

Brand
Thermo Fisher Scientific
Model
VarioskanFlash- Omnia
Type
Installation guide
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 1 of 15
Setup Guide on the Varioskan® Flash Multimode 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
Thermo Scientific Varioskan® Flash Multimode Reader
NOTE: The Thermo Scientific Varioskan® Flash Multimode 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
Thermo Scientific instruments or software, please contact Thermo Scientific at 1-800-522-7763
or www.thermo.com.
A. Recommended Optics
wavelength (nm)
diameter (nm)
Excitation
360/12 monochromator
Emission 1
485/12 monochromator
B. Instrument Setup
1. Make certain plate reader is turned on, and open up Thermo Scientific SkanIt
control software on computer.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 2 of 15
Setup Guide on the Varioskan® Flash Multimode 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 SkanIt opens, it will default to a generic starting page. Select the "New
Session" icon to open a new session and create a new protocol.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 3 of 15
Setup Guide on the Varioskan® Flash Multimode 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. At this point, a popup window will appear. Select "Empty Protocol" on the left,
and enter a session name at the bottom. Select Next when finished.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 4 of 15
Setup Guide on the Varioskan® Flash Multimode 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. Select your plate from the available list. When finished, select Next.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 5 of 15
Setup Guide on the Varioskan® Flash Multimode 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. Once the session has been named and a plate selected, click the Finish tab to
create a new protocol.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 6 of 15
Setup Guide on the Varioskan® Flash Multimode 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. A new window will appear. The next step is to define the plate layout. Select the
Wizard tab in the upper right.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 7 of 15
Setup Guide on the Varioskan® Flash Multimode 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. The Wizard popup will appear. To fill the entire plate with unknowns, enter 96 in
the "No. of unknowns" blank near the center left of the popup. In this case we
entered 36 with the filling order arrow under "Replicates" pointing left to right to
automatically select Rows A-C. Select the "Add and Close" tab when finished.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 8 of 15
Setup Guide on the Varioskan® Flash Multimode 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. The Wizard popup will close, and the plate will now be defined. As described
above, only the first 3 rows were selected for reading in this case.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 9 of 15
Setup Guide on the Varioskan® Flash Multimode 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. From the Session menu on the left, select "Protocol". The screen will change to
the one below. In the "Action" menu on the left, select the "Incubate" icon to set
the incubation temperature.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 10 of 15
Setup Guide on the Varioskan® Flash Multimode 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. Selecting the Incubate icon will cause the screen to change to look like the one
below. Select your incubation temperature as shown below.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 11 of 15
Setup Guide on the Varioskan® Flash Multimode 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. When finished, click on the "Kinetic Loop" tab, located under the "Control" menu
on the left. A new screen will appear; select your total number of reads and the
interval. In this case we set a total of 61 reads at 1-minute intervals for a one-
hour assay taking readings from t=0 to 60 minutes.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 12 of 15
Setup Guide on the Varioskan® Flash Multimode 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. Now that the incubation temperature and kinetic parameters have been set, click
on the "Fluorometric" button under the Meas. window to the left. The screen will
change to the one below. Enter your excitation and emission wavelengths, and
set your optics position. When completely finished, click on the "Execute
session" tab (small green arrow in lower left) to read.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 13 of 15
Setup Guide on the Varioskan® Flash Multimode 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 14 of 15
Setup Guide on the Varioskan® Flash Multimode 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 15 of 15
Setup Guide on the Varioskan® Flash Multimode 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 Thermo Scientific Varioskan® Flash. 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
99.00.065JAK2 Inhibitor II
99.80.0097Staurosporine
06.69Kinase
% InhibitionRate (RFU/min)Rxn Cond.
99.00.065JAK2 Inhibitor II
99.80.0097Staurosporine
06.69Kinase
% 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 = 6.6918x + 190.84
y = 0.065x + 209.79
y = 0.0097x + 245.2
100
200
300
400
500
0 102030405060
Staurosporine
JAK2 Inhibitor II
Kinase
Linear (Kinase)
Linear (JAK2 Inhibitor II)
Linear (Staurosporine)
y = 3.9902x + 202.95
y = 0.1879x + 224.28
y = -0.079x + 239.42
200
300
400
500
0 102030405060
Staurosporine
JAK2 Inhibitor II
Kinase
Linear (Kinase)
Linear (JAK2 Inhibitor II)
Linear (Staurosporine)
95.30.188JAK2 Inhibitor II
102-0.079Staurosporine
03.99Kinase
% InhibitionRate (RFU/min)Rxn Cond.
95.30.188JAK2 Inhibitor II
102-0.079Staurosporine
03.99Kinase
% InhibitionRate (RFU/min)Rxn Cond.
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Thermo Fisher Scientific VarioskanFlash- Omnia Installation guide

Brand
Thermo Fisher Scientific
Model
VarioskanFlash- Omnia
Type
Installation guide
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