My protein is UFM1 tagged with Twin-Strep tag. I double-checked and my tag is not cleaved off or hidden. I made purification with IBA kit (Tactin XT 4Flow high capacity spin kit) with subsequent Silver Stain which showed that all my protein is in Flow. I supported it with WEstern Blots, too.
My lysis buffer was: IP buffer (50 mM Tris-HCl, pH 7.4 at 4 °C, 150 mM NaCl, 1 mM EDTA) supplemented with 0.5% Non-idet P40 substitute and protease inhibitor cocktail.
I transfected A549 cells with my UFM1-2X-Strep construct and the expression was good.
The kit is new and working. I am attaching my silver stain, where first 2 lines are whole lysates before columns, next 2 are flow and then 4 lanes of washes and last 4 - my elutions.
If your protein is secreted there could be too much biotin in your medium which could prevent the binding of the Strep-tag to the matrix. You could check the composition of your medium.
It is not, I washed cells before lysis with PBS and also I used Biolock to not have biotin interference
Hi Alina,
for a better understanding, could you clarify how you have tested that the tag is present/complete/accessible? I was also wondering what you have used in your Western Blot for detection.
The most likely scenario that comes to my mind is that that the tag is not accessible in the native state of the protein. In that case, it would still be detectable in Western Blot, since there the protein is usually denatured.
It could also be possible that your protein has bound to the beads, but could not be eluted. If you protein expresses well, with the small volume of the Strep-Tactin®XT Spin column kit it might be possible that you see the bands in the flow-through because there weren’t enough beads to bind all of the protein. Have you tested if any protein has bound to the beads at all? To do that, you can take you beads after elution and boil them in SDS loading buffer. Afterwards, check the sample in an SDS gel/Western Blot.
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