Hello,
"I am observing an unusual phenomenon where the protein concentration in my flow-through sample is consistently higher than that of the lysate sample when run on gel, Still, I am obtaining a good yield of my target protein. I am using his tag, nickel resin, and running equal volumes of the lysate and flow-through samples on the gel, although the volume of the elution sample is not equal. I would appreciate any insights or ideas on what could be causing this discrepancy.
I am attaching a picture of the gel.
The first red circled band from the left is crude lysate (1 ul sampel+24 ul water), the second is flow through (1 ul sample +24 ul water), and the third is elution (15 ul sample+ 5 ul water).
You might not be using enough of the resin when eluting. It might be maxing out binding, with a large amount of protein then flowing through the beads. I recommend adding more resin or passing it through for another purification.
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