Hello guys,
I am recently doing WB to detect soluble and insoluble species of a protein using sequential extraction. I have a question about the loading control.
Generally, we use housekeeping proteins as loading controls, i.e actin. We consider it is proportional to the total protein in the lysis under the prerequisite that the solubility of actin and the target protein in the lysis are the same. However, my target protein has different solubility due to the oligomer and fibril species. The actin has the same solubility. Would it be possible that the majority of actin is dissolved in the first phase buffer, only the actin trapped in the aggregates in the pallet will be released in the SDS buffer (second phase buffer)? Thus it is not suitable to use housekeeping proteins as loading controls in this case.
I appreciate any input!
Thank you,
Waijiao
Hi Waijiao, it is acceptable to use equal total cell protein concentration as the loading control.
The same applies with SDS-soluble cell pellets. If your protein has aggregated, it will show up stronger on the WB. But you are not biasing the results because you are loading equal cell protein concentration.
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