EMSA problem - differently shifted bands (Oct/01/2007 )
I perfomed traditional EMSA with radioactive probe and purified putative transcriptional regulator.
Bands were shifted as expected, however, level of 'shifting' is somewhat wierd.
As you may see the attached figure, the band of lane 3 is more 'shifted' than the band of lane 2.
I don't think there are multiple binding sites. The difference between two lanes are protein concentration : 2-fold more protein was added in lane 3.
Do you think this result is normal? Why would these 'differently' shifted bands occur?
Thanks in advance for your replies.
I perfomed traditional EMSA with radioactive probe and purified putative transcriptional regulator.Bands were shifted as expected, however, level of 'shifting' is somewhat wierd.
As you may see the attached figure, the band of lane 3 is more 'shifted' than the band of lane 2.
I don't think there are multiple binding sites. The difference between two lanes are protein concentration : 2-fold more protein was added in lane 3.
Do you think this result is normal? Why would these 'differently' shifted bands occur?
Thanks in advance for your replies.
The EMSA looks good. As you used a purified protein I guess the band can't be a different complex. Could it have some different post-translational modification or be binding something else? What is the difference between 1, 2 and 3? Also they're not difference in salt or something else that could effect the running of the band?
Ceri
Thank you Ceri 
The difference between 1 to 3 is protein concentration: protein was serially diluted from 3 to 1.
Salt concentration might be different because dw was used for protein dilution. This might be a cause.
Also protein has phosphorylation site, so I compared the shifting from dephosphorylated form and phosphorylated form (+acetyl P).