Thermo Fisher Scientific Synergy4- Omnia Installation guide

Brand
Thermo Fisher Scientific
Model
Synergy4- Omnia
Type
Installation guide
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 1 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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
BioTek Instruments Synergy™ 4 Hybrid Multi-Mode Microplate Reader
NOTE: The BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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
Dichroic Mirror
(7138400) 400
NOTE: This document shows how to set up the reader using filters. We have also validated
this reader to perform Omnia® assays using monochromators, but it is not described here.
B. Instrument Setup
1. Make certain plate reader is turned on, and open up BioTek Gen5 software on
computer.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 2 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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 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.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 3 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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. At this point, a blank protocol will open (below). To ensure that the correct
instrument is selected, click on "Reader Configuration" under tools.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 4 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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. Check that the instrument selected is correct (below). If not, select "Delete" to
remove the existing instrument, and then "Add".
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 5 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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. 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.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 6 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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 "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.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 7 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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. 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).
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 8 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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. 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).
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 9 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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. 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".
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 10 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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. Omnia® assays are also run at 30 degrees. Select the "Set Temperature" tab
and set the reader incubator to 30ºC and click "OK".
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 11 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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. 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.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 12 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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. 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".
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 13 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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. 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).
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 14 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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
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.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 15 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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.
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.
123456789101112
JH1/JH2 A Staur. JAK2 Inh. II
BNo Inh. No Kinase 100% Phosp.
JH1/JH2 C Staur. JAK2 Inh. II
V617F D No Inh.
E
F
G
H
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 16 of 17
Setup Guide on the BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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. 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 BioTek Instruments Synergy™ 4 Hybrid Multi-Mode 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:
Rxn Cond. Rate (RFU/min) % Inhibition
Kinase 431.05 0
Staurosporine 56.903 86.8
JAK2 Inhibitor II 4.36 99.0
Rxn Cond. Rate (RFU/min) % Inhibition
Kinase 107.97 0
Staurosporine 11.165 89.7
JAK2 Inhibitor II 0.5825 99.5
Figure 2: Omnia® Kinase Assay. Omnia® assay performed as described above and
read on BioTek Instruments Synergy™ 4. Assay results monitored at 1-minute intervals,
data graphed in Microsoft Excel.
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
JAK2 JH1/JH2
V617F
y = 107.97x + 3928.2
y = 11.165x + 3801.3
y = -0.5825x + 4339.3
2000
4000
6000
8000
10000
12000
14000
0 102030405060
Staurosporine
JAK 2 Inhi bi tor II
Kinase
Linear (Kinase)
Linear (JAK2 Inhibitor II)
Linear (Staurosporine)
y = 431.05x + 5882.8
y = 56.903x + 4680.8
y = 4.36x + 3830.4
2000
4000
6000
8000
10000
12000
14000
0 102030405060
Staurosporine
JAK 2 Inhibi tor II
Kinase
Linear (Kinase)
Linear (JAK2 Inhibitor II)
Linear (Staurosporine)
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Thermo Fisher Scientific Synergy4- Omnia Installation guide

Brand
Thermo Fisher Scientific
Model
Synergy4- Omnia
Type
Installation guide
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