I have transformed and expressing his tagged-mOrange protein in BL21 strains, later I am expecting the high purity(99-100%) mOrange protein without having changed in protein configuration. So, what would be the best way to achieve such purity?
Look into using liquid chromatography, if its high volume prep chromatography..
Start with Ni-NTA affinity purification, you can find many protocols online. After this step you should already have very good purity, but if not enough you should proceed with IEX or GF, depending on sample size, identity and size of contaminants, etc. If you ahve no idea how to proceed I suggest you start with Ni-NTA and then come back with more specific questions.
Hello (@ Carlo Zambonelli) sir
Here are some specific questions for you
1. For how long I can keep protein (in -80)?
2. How to find out the concentration if the MW is unknown?
3. How to ensure the purity?
4. Right now the protein is being stored in imidazole buffer (pH 7.4). Will the concentration change if I change the buffer? If so? How much will it decrease?
5. If the pH of the new buffer is 10 and I store the protein in it. Will the protein gonna survive ? or it may degrade the protein with due course of time.
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