Is there a way I can lyse cells with a GFP-construct in a mild RIPA buffer, put the lysate into a 96 well plate, and quantify the GFP fluorescence? I want to quantify the relative GFP expression between different constructs.
You should not use ELISA plates for quantification of fluorescence. ELISA plates are transparent and are meant for OD measurement. You should use opaque black Nunc plates specially designed for the purpose. Unlike transparent ELISA plates these plates are opaque and thus there is no cross-talk of signals between two wells. Plus, these reflect better. I have experience of adding lysis buffer to to 96-well cell culture plate, picking up contents of each well and transferring to a Nunc-plate, addition of substrate for measuring firefly luciferase expression. Similar, method may work with GFP, perhaps. Good luck!
http://www.sigmaaldrich.com/catalog/product/sigma/p8616?lang=en®ion=PK
https://worldwide.promega.com/resources/pubhub/which-plates-to-choose-for-fluorescence-and-luminescence-measurements/
http://www.fishersci.com/ecomm/servlet/fsproductdetail_10652_1679871__-1_0
Do you necessarily want to lyse the cells to do this? you can measure fluorescence directly from plated cells, using either a global well fluorescence measurement - see for example Bronwyn J. Green and John E.J. Rasko (2002) Human Gene Therapy 13:1005-1013. You can now use quad monochromators (i.e. dual for excitation and dual for emission to get a really good spectral resolution - e.g. available on the Enspire from PerkinElmer) to achieve this. And the new Ensight Reader (PerkinElmer, http://www.perkinelmer.com/ensight/) can also measure at the cellular level, providing statistics on the spread of the level of expression per cell. You may look in your lab to see if you have any instrumentation that would enable you to do one of these 2 types of readings. Good luck.
The MST (MircoScale Thermophoresis) instruments are very sensitive in fluorescence detection. So you could "misuse" them to just measure fluorescence intensity of your cell lysate. The measurement is taking place in single-use capillaries, so you do not have to care about washing, equilibration or cross-contamination.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA