I am now doing EMSA with biotin linked probe, however, I can't see any unbonded free nucleotide probe. Is this normal? Should I shorten the time of electrophoresis？
Here is my results. Probe was labeled with biotin and visualized with HPR conjugated streptavidin.
Hi;
What is the percentage of the Gel you have used for EMSA:
Try to check the probe of Single strand and annealed form of the probe in NATIVE-PAGE stained with EtBr.
I suggest you to check the the probe concentration because In your gel I could see the probe concentration is not Unique.
The probe may be running off the gel due to its small size. If you covalently label it with a fluorescent dye instead of biotin, you can observe its behavior during electrophoresis to see where it is on the gel. That would also save you the trouble of blotting.
You might be able to stain it after electrophoresis, although it might stain poorly with ethidium. Try using SYBR Green or RiboGreen instead.
Kannan Balakrishnan 6% strength of the gel and annealed probes were used in my assay. Do you also suggest that the free probes were out of gel? Should I try to reduce the concentration of the protein?
Guopeng Miao I suggest you to run 8% gel and the free probes running out of the gel so try to run the gel based on the loading dye separation.
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