If the target DNA sequence can have binding specificity with any other random protein present in the lysate, then how to nullify this fact. Though, I have tried this EMSA with purified protein, now I am aiming to see the effect of mutant on DNA binding ability, thats why want to try with lysate.
I could imagine that you will obtain smears instead of sharp bands on your EMSA gel if you are using cell lysate...
You could try MicroScale Thermophoresis instead. You order your DNA sequence with a fluorescent label attached (Cy5 for example). Then you add cell lysate of your bacteria with overexpressed target protein and as a control you could use cell lysate of bacteria transformed with an empty plasmid.
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As I can understand from your question, you need to do a pull down assay to identify proteins that bind to your sequence. Since a lysate would contain many non specific DNA binding proteins you may no get a meaningful result in an EMSA. Please explain your problem in detail if I didn't answer your question fully.
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