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We know that when performing cell-based assays, cell culture requirements can slow the whole process down. Maintaining the cells in culture, harvesting and plating require a lot of time, not to mention the reagents and plasticware you need. Though we can’t take the steps away we can make the process more efficient.
Maximize your research potential - measure multiple targets in the same sample.
Given such a lengthy process, it makes sense you’d want to extract as much information as possible from each culture well. While these types of experiments usually involve the purchase of specialized reagents and detection equipment, our AlphaScreen® SureFire® suite of endogenous phospho-protein kinase detection assays only need a small amount of lysate per assay well – typically less than 5 mL ─ plus no transfection is required.
Figure 1
MCF7 cells were seeded, in a 96-well CulturPlate at a density of 50 K per well overnight in media containing 10% serum. The following morning, the media was replaced with media containing various concentrations of UCN-01, a promiscuous kinase inhibitor. After the cells were incubated with UCN-01 for 3 hours, the media was removed and the cells were lysed with 50 mL 1X lysis buffer. 4 mL aliquots of lysate were transferred to the wells of a 384-well ProxiPlate and analyzed for either phospho-ERK, phospho-BAD, phospho-GSK3b or phospho-p70S6 kinase, using standard AlphaScreen SureFire protocols. Data demonstrates that in MCF7 cells, UCN-01 has an inhibitory effect on the basal phosphorylation of BAD, GSK3b and p70S6 kinase, but at high concentrations has a stimulatory effect on ERK phosphorylation over the experimental timeframe.
Figure 2
MCF7 cells were seeded, in a 96-well CulturPlate at a density of 50 K per well overnight in media containing 10% serum. The following morning, the media was replaced with media containing various concentrations of rapamycin, a specific mTORC1 inhibitor. After the cells were incubated with rapamycin for 3 hours, the media was removed and the cells were lysed with 50 mL lysis buffer. 4 mL aliquots of lysate were transferred to the wells of a 384-well ProxiPlate and analyzed for either phospho-ERK, phospho-BAD, phospho-GSK3b or phospho-p70S6 kinase, using appropriate AlphaScreen SureFire protocols. Data demonstrates that in MCF7 cells, rapamycin has an inhibitory effect on the basal phosphorylation p70S6 kinase, which is directly phosphorylated by the mTOR complex 1, but has no effect on basal BAD, GSK3b or ERK phosphorylation over the experimental timeframe.
One cost effective solution.
By using AlphaScreen SureFire kits, a 96-well or 384-well cell culture can be assayed for several targets. Information regarding specificity across major cellular pathways, or at multiple nodes within a particular pathway, can be obtained from a single well. The bottom line – our range of AlphaScreen SureFire assays affords your lab an easy and inexpensive way to optimize your cellular experiments.
To learn more about our portfolio of AlphaScreen SureFire® assays, please visit our Web site or contact a PerkinElmer sales representative. As always, we’re looking forward to working together.
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PerkinElmer Tools for Kinase Screening
Detection Instruments
Automated Liquid Handling
Microplates
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Attend these PerkinElmer special events held in conjunction with SBS/ELRIG and SBS Cell-based Assay
Don't miss our Kinase Application Group
Monday, September 22nd - 2pm to 6pm at the Bournemouth Hillcliff Marriott Hotel. This is a pre-event meeting to the SBS/ELRIG event in Bournemouth, UK September 23-24.
Our Speakers
- Doriano Fabbro from Novartis, Basel, Switzerland presents “Everything you wanted to know about targeting the kinome”
- Michael Crouch from TGR Biosciences presents “Multiple phosphoprotein target analysis and profiling with AlphaScreen SureFire® Cellular Kinase Assays”
- Noushin Mirjalili from Cerep SA presents “Kinase assay development, a case study adapting assay development criteria to meet various outsourced research objectives.”
- Collette Chitty and Roy Katso, from GlaxoSmithKline present "A homogenous mechanistic cellular assay for identification of small molecule inhibitors."
After the meeting, a wine tasting with lecture by James Manson is scheduled from 6pm to 8pm, also at the Marriott Hotel. Click here for more details or to preregister.
Wednesday, October 22nd - 9am - 4pm at the Radission Valley Forge Hotel. This isa pre-event meeting to the SBS Cell-based Assay Symposium event in King of Prussia, PA USA Click Here for more details on this applications group.
PerkinElmer Tutorial
SBS Cell-based Assay Symposium King of Prussia, PA USA
Thursday, October 23rd - 3:35pm - 4:20pm, Conestoga Meeting Room
Aequorin versatility: bright AequoScreen® cell lines run in luminescence mode on a FLIPRTETRA® and their characterization in a glow-type assay
Vincent Dupriez, Ph. D., Senior Scientist, Cellular Sciences, PerkinElmer, Inc.
The bright signal of aequorin cell lines enables reading on a wide range of instruments in different detection modes. Data will be presented to demonstrate the flexibility of PerkinElmer AequoScreen® (Aequorin) cell lines in both flash and glow luminescence.
Click here for more details or to register.
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