Thermo Fisher Scientific PHERAstar- Omnia Installation guide

Brand
Thermo Fisher Scientific
Model
PHERAstar- Omnia
Type
Installation guide
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 1 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: drugdiscoverytech@invitrogen.com
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.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 2 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: drugdiscoverytech@invitrogen.com
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.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 3 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: drugdiscoverytech@invitrogen.com
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.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 4 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: drugdiscoverytech@invitrogen.com
4. A new window will pop up. Select “Fluorescence Intensity” and “Endpoint” and
then select “OK”.NEED PIC FOR PHERAstar here
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 5 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: drugdiscoverytech@invitrogen.com
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.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 6 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: drugdiscoverytech@invitrogen.com
6. Select the wells you wish to read. When finished, select OK.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 7 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: drugdiscoverytech@invitrogen.com
7. You will return to the initial settings window. From the drop-down menus at the
top, select "Measure" and "Measure" again.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 8 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: drugdiscoverytech@invitrogen.com
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.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 9 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: drugdiscoverytech@invitrogen.com
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.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 10 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: drugdiscoverytech@invitrogen.com
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.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 11 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: 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 12 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: 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 13 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: drugdiscoverytech@invitrogen.com
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 + 235.46
200
250
300
350
400
450
500
0 102030405060
Staurosporine
JAK 2 Inhi bi to r 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 to r 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 to r 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
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 to r 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 to r 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 to r 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 = 1070.1x + 14357
y = -21.626x + 17243
y = -6.0781x + 17387
2000
12000
22000
32000
42000
52000
62000
0 102030405060
Staurosporine
JAK2 Inhibitor II
Kinase
Linear (Kinase)
Linear (JAK2 Inhibitor II)
Linear (Staurosporine)
y = 8079.7x + 50716
y = -44.494x + 37321
y = -28.271x + 47540
2000
22000
42000
62000
82000
102000
122000
142000
0 102030405060
Staurosporine
JAK2 Inhibitor II
Kinase
Linear (Kinase)
Linear (JAK2 Inhibitor II)
Linear (Staurosporine)
102.3-21.63JAK2 Inhibitor II
100.6-6.08Staurosporine
01070.1Kinase
% InhibitionRate (RFU/min)Rxn Cond.
102.3-21.63JAK2 Inhibitor II
100.6-6.08Staurosporine
01070.1Kinase
% InhibitionRate (RFU/min)Rxn Cond.
100.6-44.44JAK2 Inhibitor II
100.3-28.27Staurosporine
08079.74Kinase
% InhibitionRate (RFU/min)Rxn Cond.
100.6-44.44JAK2 Inhibitor II
100.3-28.27Staurosporine
08079.74Kinase
% InhibitionRate (RFU/min)Rxn Cond.
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Thermo Fisher Scientific PHERAstar- Omnia Installation guide

Brand
Thermo Fisher Scientific
Model
PHERAstar- Omnia
Type
Installation guide
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