I am getting smears in EMSA (electrophoretic mobility shift assay) result. what should i do to show proper binding instead of smear??

Check sample quality: Ensure that your sample is of good quality and is not degraded, as this can cause smearing.

Optimize the binding reaction: Ensure that the binding reaction conditions (e.g. protein and DNA concentration, incubation time, and temperature) are optimized to allow for proper binding to occur.

Increase the specificity of the binding reaction: Use specific competitor DNA or a cold probe to increase the specificity of the binding reaction and reduce background noise.

Check for non-specific binding: Ensure that there is no non-specific binding occurring, which can cause smearing. This can be done by using a control reaction with a non-specific competitor DNA or by performing the reaction in the presence of a non-specific binding inhibitor.

Adjust electrophoresis conditions: Ensure that the electrophoresis conditions (e.g. gel concentration, running buffer, and voltage) are optimized to reduce smearing and allow for clear separation of the protein-DNA complexes.

Repeat the experiment: If smearing persists, repeat the experiment using fresh samples and reagents to rule out any technical errors.

Shubham Deshmukh

Hi Shradha,

I too had this issue when I did EMSA with protein and Cy5-labelled RNA and managed to solve this issue partly.

First of all. Although most EMSA results that are published do not have smears, smearing may not be bad. Smearing may mean at least two things in my opinion. First, your protein converts into a multisubunit form as a result of binding to DNA. If this happens, there may exist multiple species of protein-DNA complexes resulting in a smear and protein getting stuck in wells. Second, your DNA may have multiple binding sites for your protein. Some sites are occupied and some are not, hence multiple species of protein-DNA complex and a smear and protein getting stuck in wells.

I solved this issue partly (smears reduced to some extent, but not fully), by first incubating protein and RNA followed by UV crosslinking and brief heating in LSB. Please see the two images I attached. It is clear that image 1 has less smear and no protein stuck in wells. Image 2 is before using my trick. The idea was to covalently link protein-RNA complexes by UV and treating with SDS to remove multiple subunits of protein if they exist.

UV mainly covalently links protein-DNA/RNA as opposed to protein-protein.

If you need more help, please email me at [email protected]

Nikolay Klyashtornyy

Here are a few suggestions to improve your EMSA results:

Optimize protein concentration: Try different protein concentrations to find the optimal amount that yields a strong and specific DNA-protein complex. Too little protein will result in weak bands or smears, while too much protein can cause non-specific binding and smears.

Increase incubation time: Sometimes, a longer incubation time can result in a stronger and more stable DNA-protein complex. Try increasing the incubation time gradually and see if it improves the result.

Use different buffer conditions: Try using different buffer conditions such as pH, salt concentration, and detergent to optimize the binding between the protein and DNA. Changing the buffer conditions can affect the stability of the protein-DNA complex.

Add competitor DNA: Adding an excess of non-specific DNA, such as poly(dI-dC), can help reduce non-specific binding and improve the specificity of the protein-DNA complex.

Use a different gel system: If reducing the percentage of the gel did not work, try using a different gel system, such as a native gel, to resolve the DNA-protein complex.

Test different probes: Consider testing different probes to ensure that the probe used is specific for the DNA-protein complex you are trying to study.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

Shradha Surin

thank you Nikolay Klyashtornyy for your suggestions. I was already using a competitive DNA inhibitor and I tried eliminating it also. Also i did change the pH of buffer. But all these changes didn't yield any difference in my results. Increasing the incubation time and also changing the gel percentage of my native gel did somehow improve my results. Probably varying the protein concentration may be helpful. I shall be trying that soon

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