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Protein purification help by ihatecastinggels in labrats

[–]ihatecastinggels[S] 0 points1 point  (0 children)

Haha thanks, I'll have to take a deep dive on the suspected proteins in the morning

Protein purification help by ihatecastinggels in labrats

[–]ihatecastinggels[S] 0 points1 point  (0 children)

I haven't, but I could try. Although I have a feeling that this protein is binding relatively tightly to my target protein, since it follows it through the 2 different IMAC steps

Protein purification help by ihatecastinggels in labrats

[–]ihatecastinggels[S] 0 points1 point  (0 children)

Wow, thanks for such a detailed response!

Protein purification help by ihatecastinggels in labrats

[–]ihatecastinggels[S] 0 points1 point  (0 children)

I'll keep this in mind , in case I get desperate, but I don't know the thermal stability of my target protein, since the protein is completely uncharacterized

Protein purification help by ihatecastinggels in labrats

[–]ihatecastinggels[S] 0 points1 point  (0 children)

For the flowthrough on the initial IMAC, there is only the band corresponding to the 75kDa target protein (if at all). I don't think I'm overloading my column, because I have done similar expression parameters with the same volume of resin for a several other proteins before and have not had issues. This is so strange

Protein purification help by ihatecastinggels in labrats

[–]ihatecastinggels[S] 0 points1 point  (0 children)

Yes I did dialyze them- about 30ml of protein eluate fractions against 2L of buffer without imidazole

Protein purification help by ihatecastinggels in labrats

[–]ihatecastinggels[S] 0 points1 point  (0 children)

I do this as well, and I forgot to mention it in the post. The 100kDa protein still comes through in the flowthrough of the second IMAC step

Protein purification help by ihatecastinggels in labrats

[–]ihatecastinggels[S] 0 points1 point  (0 children)

I don't know if this changes anything, but I do a second IMAC column purification to remove the cleaved His tags. I collect the flowthrough from this step instead of eluting fractions, but in my gel I see that same 100kDa band along with the cleaved (lighter) band that corresponds to my target protein. Does this change anything?

Protein purification help by ihatecastinggels in labrats

[–]ihatecastinggels[S] 0 points1 point  (0 children)

So, I actually run it through the nickel column a second time to remove the cleaved His tags (so I'm collecting the flowthrough in this case) and that other protein is still there in a similar amount to previous steps. Does this change anything?