I am trying to measure the Fluorescence intensity of my gel using a microplate reader but I'm not sure which reading, top or bottom, to trust?
the results of top reading and bottom reading are quite different.
Hi.
If you are using a protein sample, top reading will be more sensitive. On the other hand, while using cell based assays where cells adhere to the bottom of the cell, bottom reading will be better. Again, for bottom reading, the thickness of your microplate should be minimum.
Hi Mahshid Pashootan , I think your situation is slightly different from using a regular 96 well plate in a micro plate reader. Are you here mentioning about a gel like SDS-PAGE. In that case the thickness of your gel would matter for your reading from the bottom. Also, possible light scattering might affect the top reading. In a situation like this, it will be decision to make which method will be more sensitive with minimum interference. When first thing you may need to do is to record the emission spectra of your material from both top and bottom reading. Then compared it with the emission spectra of the fluorophore (possibly can find from the literature) to compared the emission profiles from both top and bottom values. From this data you will have an a idea about which method is more reliable in your case. Just having a larger response doesn’t necessarily make one reading method is better. It is always important to to make sure you have minimum interference for you emission.
Hope this information helps.
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