4 replies to this topic

[sata]

Hello,
Is there ayone working on EMSA.I am new to it.I have problems with my results.I see smear after revelation of film.I am using Biotin kit.There is high backround.Can anyone help me?

[rimal]

are you using nitrocellulose membrane or PVDF?

[sata]

rimal, on 20 December 2010 - 11:38 PM, said:

are you using nitrocellulose membrane or PVDF?

I m using nitrocellulose membrane but still i don't see any DNA-protein binding complex.Its just like a smear.I have checked for protein degradation also but western blot is fine so it means that proteins have not been degraded.If u have any suggestions plz do tell me.
Thanks

[jennyal]

sata, on 21 December 2010 - 12:23 AM, said:

rimal, on 20 December 2010 - 11:38 PM, said:

are you using nitrocellulose membrane or PVDF?

I m using nitrocellulose membrane but still i don't see any DNA-protein binding complex.Its just like a smear.I have checked for protein degradation also but western blot is fine so it means that proteins have not been degraded.If u have any suggestions plz do tell me.
Thanks

Hi!

A smear can indicate that you have loaded to much proteins onto the gel. Try and reduce the amount of nuclear proteins you use in the EMSA reaction. I recommend usig 0,5 micrograms for a 20 microL reaction as a starting point then go from there. Do you use your own nuclear extract or have you bought it? It could be that your extracts are not clean enough if you have done your own nuclear protein extraction. Measure the conc if you make your own.
Good luck!

[sata]

jennyal, on 16 March 2011 - 04:37 AM, said:

sata, on 21 December 2010 - 12:23 AM, said:

rimal, on 20 December 2010 - 11:38 PM, said:

are you using nitrocellulose membrane or PVDF?

I m using nitrocellulose membrane but still i don't see any DNA-protein binding complex.Its just like a smear.I have checked for protein degradation also but western blot is fine so it means that proteins have not been degraded.If u have any suggestions plz do tell me.
Thanks

Hi!

A smear can indicate that you have loaded to much proteins onto the gel. Try and reduce the amount of nuclear proteins you use in the EMSA reaction. I recommend usig 0,5 micrograms for a 20 microL reaction as a starting point then go from there. Do you use your own nuclear extract or have you bought it? It could be that your extracts are not clean enough if you have done your own nuclear protein extraction. Measure the conc if you make your own.
Good luck!

Hello.Thanks for replying>Now I have got rid of that smear but there is another prblem as the competition reaction is not working.I have used 200x of competitor but still no difference.Can u suggest anything in this regrad.