I want to measure the solubility of some proteins expressed in E.coli, and while reading former reports, they used beads to grab the his-tagged proteins, measured the content as "total", then centrifuge, and measured the protein concentrate in supernate as "soluble". However, I wonder that insoluble proteins(for example, incorrectly folded ones) may not efficiently bind to the affinity beads, so a part of it may lost in the purification step, making the soluble: insoluble ratio in "total" biased.
If you know your total protein, you can use your soluble fractions to deduce the insoluble fraction in each sample.
When your protein is insoluble you get it in the form of an aggregate, which will be lost in centrifugation. The protein is not refolded and the his-tag is most of the time inaccessible to affinity beads or antibodies efficiently.
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