EMSA total or nuclear extract - (Jul/07/2007 )
hi!
I have a question about EMSA. Is it possible to use total protein extract for supershift experiment? I was trying to use nuclear extract but cant get good data? I think that I have to use more extract, but do not have that anymore? Thank you
-laska-
QUOTE (laska @ Jul 7 2007, 10:18 AM)
hi!
I have a question about EMSA. Is it possible to use total protein extract for supershift experiment? I was trying to use nuclear extract but cant get good data? I think that I have to use more extract, but do not have that anymore? Thank you
I have a question about EMSA. Is it possible to use total protein extract for supershift experiment? I was trying to use nuclear extract but cant get good data? I think that I have to use more extract, but do not have that anymore? Thank you
I think that the answer would depend on which transcription factor you plan to study. For example, if you are interested in NF-kB, you wouldn't get good results since p65 is complexed in the cytoplasm with IkB proteins. Also, you would dilute the transcriptionally active p65 which will be found primarily in the nucleus with cytoplasmic proteins. First I would troubleshoot your protocols if adding more nuclear extract doesn't work. Make sure you are getting good nuclear extracts and be sure that whatever protein you are studying is signaling during your experimental conditions. Then I would modify some of the EMSA conditions, such as salt concentrations and incubation times.
-mateo-