Posted 14 October 2003 - 08:24 AM
Has anyone had any experience with ELISAs using immfluorescence. I'm using a biotynlated anti with an Alexa Fluor 488 strepavidin conjugate (10 ug/ml), problem is doesn't work even in biotin control wells. Any ideas welcome.
Posted 04 November 2003 - 10:21 AM
Fluorescence may not be a good idea since many clear plates have crosstalk between wells and reflective properties. You'll have to use black opaque plates with optically clear bottoms. Alternatively, if you have a top reading device, you could use plates with black wells--I don't know if any of these are capable of binding peptide/protein. The read time of most devices also seems short. I don't know if the fluor can be adequately excited and measured in the short time.
Finally, it seems that your 488-SA is very high. I think 1 microgram/ml should be enough. You can test your machine for linearity/readout efficiency (and your plates for crosstalk) by diluting 488SA in PBS in replicates of 3.
Posted 04 November 2003 - 10:25 AM