alternative technique for EMSA - (Apr/11/2007 )
Hello everyone,
Does someone knows an alternative for EMSA technique? Also does someone can give me an idea of time/cost of an EMSA technique? Some people said one year, others 14 days. We want to know which complex of transcription factors of the AP-1 family binds with the promoter. Could streptavidin beads been an alternative for EMSA? Is it possible to bind the synthesised cDNA to this beads, mixed with nuclear extract and than analysed on a western blot?
Can anyone give me some hints in finding an alternative for EMSA? 
Many thanks in advance 
Indra
I've had great success with the lightshift kit from pierce. I took me about 2 weeks to optimize the binding reactions and another 2 to complete the study. My probes were 50bp and biotin labeled; ordered from mwg but I'm sure other companies will do the same. Do you know the region that binds the transcription factor, if not maybe some bioinformatics is need first? EMSA can only be done with short DNA fragements not whole promters. Have you used any other approaches e.g. gene-reporter assays in conjunction with transfection of transcription factors?
As far as identifying the transcription factor EMSA alone will not tell you much but if you know it is of the AP1 family then you could try supershift assays using specific antibodies or use competitor probes of known motifs.
Hope this helps
As far as identifying the transcription factor EMSA alone will not tell you much but if you know it is of the AP1 family then you could try supershift assays using specific antibodies or use competitor probes of known motifs.
Hope this helps
Many thanks for your reply.
We would synthesise a cDNA labelled with biotin in the assumption to bind this on a beads coated with streptavidin. The size of the cDNA is 30bp. We take the region of the AP-1 with a few bp extra before and after. We used already a TransAM technique from Active Motif based on a coated Elisa with transcription factor. There were a few candidates but we want to know which of the seven forms a complex to bind with the promoter. This we want to analyse with EMSA. But because my time is short (1 month left) I’m searching for a quick alternative.
Is it possible that you send me your protocol?
Again, many for helping!!
The manual for the kit can be found at this link: http://www.piercenet.com/files/0919as4.pdf. I also used their binding reaction kit which contains various additives mg, glycerol etc. This can probably substituted with what you already have in your lab. The exact conditions will need to be determined experimentally. The probes should be double stranded; the biotin is used for detection procedure not for linking to beads.
You should also think about the source of the nuclear extracts, presumably a cell line but native tissue can also be used. If possible extracts should be used from a source which expresses your protein of interest. I'm not sure that EMSA will actually give you the answer you are looking for. By the sounds of it you already know that which transcription factors bind to the promoter. Are you just trying to narrow down the region? You will have to use specific antibodies against transcription factors in a 'supershift' assay to determine which proteins are forming complexes. If you don't have any good candidates this might not be a good approach.
feel free to contact me if you want more info