I am using an EMSA experiment to determine the core element of protein binding sites using DIG gel shift assay kit from ROCHE. I have obtained a good quality shifting of the band which was not observed in the lane loaded with probe only. But there was no change in the intensity of the band even after the addition of unlabeled competitors with a molar excess of 50 to 500 . The Nuclear extract used was 6 micrograms, probe concentration 120 fmol and nonspecific competitor (poly dI-dC) 1 microgram. Binding was performed in room temperature for 35 min (5 min with poly (dI-dC), 15 min with competitor and then 15 min with probe). Could someone please suggest what to do, how can I overcome this problem?
Article Characterization of DNA-Binding Proteins Using Multiplexed C...
Then probably protein binding is not specific:
"Binding competition analysis: If competitor DNA is identical to and in relatively large excess over the labeled DNA, >90% of the signal should be eliminated from complexes corresponding to protein(s) that interact with the binding-site DNA in a sequence-specific manner. Complexes unaffected or only slightly affected by the addition of competitor are thought to arise from nonspecific, low-affinity binding by abundant proteins that are present in excess to binding-site DNA."
http://www.springer.com/biomed/human+genetics/book/978-1-60327-014-4
If there is non-specific binding that could mask your real interaction, maybe you could try increasing the amount of poly dI-dC. For crude extracts up to 5 micrograms can be used. Another possibility is reducing the amount of protein added.
I think you put too much binding proteins in the assay. Did you do any gradient to determine the affinity? It is better to use the minimal protein concentration at which the probes are completely shifted.
Hey Vivek
what is your competitor? is it same probe which is being labelled?
Try reducing the concentration of protein that you are using for assay.
Use purified unlabelled probe. also when you are making the reaction, incubate with unlabelled probe for few minutes before adding labelled probes. this is just to figure out what is going on and what are the right concentration that you should use.
if you are using same fragment as unlabelled probe that is labelled to use as probe, intensity of the band should go down even if that is non-specific binding. you have to optimize these parameters. i would suggest very high concentration of unlabelled probe with different amount of protein. and once you find the protein amount that is working, then use increasing concentrations for unlabelled probe. reducing the amount of labelled probe is another option.
all the best
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