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Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 1 of 10
Setup Guide on the BMGLABTECH OPTIMA 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
BMG LABTECH OPTIMA Microplate Reader
NOTE: The BMG LABTECH OPTIMA 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 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
Filters
Excitation
360
(or similar) *contact BMG LABTECH
Emission 1
485
(or similar) *contact BMG LABTECH
B. Instrument Setup
1. Make certain plate reader is turned on, and open up OPTIMA Control software
on computer. Insert plate into plate reader.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 2 of 10
Setup Guide on the BMGLABTECH OPTIMA 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 OPTIMA Control software opens, if you do not have a pre-existing protocol
for Omnia®, 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 10
Setup Guide on the BMGLABTECH OPTIMA 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 new screen will open (below). Click on the “Show all test
protocols” 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 10
Setup Guide on the BMGLABTECH OPTIMA 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. A new window will pop up. Select “Fluorescence Intensity” and "Plate mode
(slow kinetics)"” and then select “OK”.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 5 of 10
Setup Guide on the BMGLABTECH OPTIMA 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. A new Protocol window will open automatically. Enter a test name, select plate
type, and select the Optic type. Next, set the number of flashes per well and
select the excitation and emission filter from the drop-down tabs. In the Kinetic
window, enter 61 cycles, 10 flashes per well and cycle, and 60-second intervals
to prepare a kinetic assay of 1 hour with readings taken every 60 seconds.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 6 of 10
Setup Guide on the BMGLABTECH OPTIMA 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 "Layout" tab at the top of the window. Select the wells you wish to
read by dragging over them. When finished, select "OK.
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 7 of 10
Setup Guide on the BMGLABTECH OPTIMA 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 window will appear. Select the wells you wish to read by highlighting
them. When finished, select "OK"
Omnia® Compatible Microplate Reader
Documentation
Version No.:
10 Mar 09 Page 8 of 10
Setup Guide on the BMGLABTECH OPTIMA 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 9 of 10
Setup Guide on the BMGLABTECH OPTIMA 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 10 of 10
Setup Guide on the BMGLABTECH OPTIMA 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 BMG LABTECH OPTIMA. 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)
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
90.8480.53JAK2 Inhibitor II
100.14-1.31Staurosporine
0878.81Kinase
% InhibitionRate (RFU/min)Rxn Cond.
90.8480.53JAK2 Inhibitor II
100.14-1.31Staurosporine
0878.81Kinase
% 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 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 = 379.58x + 4968.1
y = -6.9763x + 5562.3
y = 13.784x + 4915.1
2000
7000
12000
17000
22000
27000
32000
0 102030405060
Staurosporine
JA K 2 Inhi b i tor II
Kinase
Linear (Kinase)
Linear (JAK2 Inhibitor II)
Linear (Staurosporine)
96.3713.78JAK2 Inhibitor II
101.84-6.98Staurosporine
0379.58Kinase
% InhibitionRate (RFU/min)Rxn Cond.
96.3713.78JAK2 Inhibitor II
101.84-6.98Staurosporine
0379.58Kinase
% InhibitionRate (RFU/min)Rxn Cond.
y = 878.81x + 11022
y = 80.526x + 8001.2
y = -1.3121x + 8738.3
2000
7000
12000
17000
22000
27000
32000
0 102030405060
Staurosporine
JAK2 Inhibitor II
Kinase
Linear (Kinase)
Linear (JAK2 Inhibitor II)
Linear (Staurosporine)
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