Lanthascreen Assay Setup on the Perkin Elmer Envision

LanthaScreen™ Assay Setup on the Perkin Elmer EnVision

Technical Support

Invitrogen Drug Discovery Solutions: 800-955-6288, select #3, then extension 40266 Perkin Elmer Technical Support: 800-762-4000

A. Filter ordering information

Excitation filter (340/60 Perkin 2100-5010 Lance/Delfia) Elmer Emission Filter Donor (495/10, size=15mm) AND PV003 Emission Filter (set of both Acceptor (520/20, Chroma filters). Size = size=15mm) 15mm Dichroic, Lance Dual Perkin 2100-4160 (D400/D640) Elmer

B. EnVision Setup

NOTE: Before beginning, you will need to install the correct filters in an appropriate slot. The slot number is not critical but the filters do need to be next to each other for the dual read. If they are not, the machine will give you an error message and you can simply switch them.

1. Open up Wallac EnVision Manager.

2. In the Navigation tree area, scroll down to “Mirrors” and make sure that you have the “Lance Dual” mirror. If not, this will need to be purchased and installed. See the Appendix for ordering information.

3. In the Navigation Tree area, scroll down to “Filters, excitation”. Make sure that you have the UV (TRF) 340 excitation filter. 4. In the Navigation Tree area, scroll down to “Filters, emission.” Select “New” and enter your Chroma 495 filter as shown in the following screen shot: Note that the barcode may differ, depending on the filter holder you are using. 5. In the Navigation tree area, choose “Emission” under “Filters” and enter your Chroma 520 emission filter as shown in the screenshot below: Note that your barcode number may be different depending on the filter holder you are using.

6. In the Navigation Tree area, scroll down to Lance – Time Resolved FRET. This will allow you to set up a Time Resolved Assay. 7. Select “New” from the Menu bar. A blank label page will show up. Enter the information as it appears in the screen shot below.

The order of filters is important: the first emission filter should be the 520 and the second should be the 495. See Appendix A for ordering information. 8. In the Navigation Tree area, scroll down to “Protocols.’’ Select “New” and enter an “ID” for the Protocol (i.e. LanthaScreen). The ID tab should be the default tab selected when you set up a new protocol. 9a. Click on the “Plate” tab and choose the appropriate plate. We recommend Corning #3676 Low Volume Non-binding 384 well plates. 9b. The screenshot below shows the plate definition for these plates. This can be added in the “Plate” section of the Navigation Tree. 10. Click on the “Operation” tab and Choose the appropriate label to be used with this protocol. In this case, choose “LanthaScreen.”

11. We have found that for this assay and this plate, height = 7 mm as defined in the Label in step #7 above works quite well. If you are using a different plate or assay volume different from 20µl, you will need to use the Optimization Wizard.

An optimization for the plate height can be done at this time by clicking on the “Optimize” button. Follow the Wizard to complete the optimization.

12. Click “Save” to save your protocol.

13. Click on “Start”. The following error message may appear because you are using the Lance Dual mirror. You can click on “Yes” and the assay should begin. 14. While the EnVision is reading the plate, you may look at data in a couple different ways. Click on the “Grid View” tab and your data could look like this: This is an actual read from the 520nm signal from a Crosstide (A1-C17) and TK (D1-F17) competitor titration. Column 18 is buffer only. 15. The data for the 495nm signal looked like this: Note that the 495 signal does not change nearly as much as the 520 signal. This is normal for this particular assay.

15. Note that the reader is prompting you to hit the “Stop” button when the read is complete. Click on “Stop.” 17. To view results, click on “Results” under your protocol in the Navigation Tree area. Results can be exported at any time to Excel by clicking on the “Tools” menu and using the “Results Export” command. Remember to use the “.csv” extension on the end of your file name.