We are purifying core Streptavidin (STA). We can obtain the streptavidin from inclusion bodies without major problems but we have difficulties to bind the STA to a Hitrap Q column, even at pH 9.2 (w/o NaCl).
It is published that it should bind the column at pH 8.6. https://www.ncbi.nlm.nih.gov/pubmed/12963352
If we don´t bind the STA to the anionic column, we can obtain it without any other protein contaminants but it appears to have some DNA contamination (high 260/280 UV absorption).
Any ideas?
Thanks!!
Juan
If the refolded streptavidin is in the form of soluble aggregates, then it may not bind well to columns. Aggregation could also be responsible for a high 260/280 absorbance because of light scattering. You can use dynamic light scattering to check for protein aggregation.
Hi Juan, the reference you gave was:
"Dialysis strategies for protein refolding: preparative streptavidin production." I don't have access to the whole article.
But can you give more details, like how you are assaying for STA? Are you looking for monomers, dimers or functional tetramers?
Dear Adam,
Thanks for your response but I think aggregation is not our problem.
Our STA is in a tetrameric state after concentration and gel filtration.
In addition, it has no UV absorption in the 300-400 nm range before concentration,
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