I have a custom HEK cell line that produces my protein of interest (8 kDa) and I want to purify the protein out of it. From my readings, there are quite a lot of steps and that will not get it to high purity. The options I read are some combination of salting out, SEC, spin filters, etc. Does someone have a suggestion for which techniques to follow and in what order to achieve a decent purity in an efficient manner? I will likely only be working with 50-100 mL of media at a time. I also have access to ultracentrifuges as needed.
Using TCA-DOC protein precipitation method. Have a look to the following. It might be helpful.
"An Optimized Approach to Recover Secreted Proteins from Fibroblast Conditioned-Media for Secretomic Analysis"
A secreted protein in cell culture media represents a very small amount in terms of mass, compared to the proteins in the serum or culture media. From 50-100 ml, you are probably in the nanogram range or less. Any kind of TCA precipitation from media will not get a pure product. Unless you have some. kind of affinity purification method, an epitope tag or antibody against your protein, this will be difficult.
Hi Connor
what do you intend to do with the purified protein? TCA will denature the protein! Given that your protein is smaller then most of the other proteins you find in cell culture supernatant, size exclusion chromatography will do a good job. However, 50-100 ml is to high a volume to load a column. What is the pI of your protein? A good thing as an initial step in the purification is to pull down your protein on anion or cation exchange beads.
Follow this by SEC and a third step as required.
Jürgen
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