Hello researchers,
I am planning to express a protein with non natural AA. When I previously expressed the protein, I found the protein expression to be very low, based on research I found the concentration of 4 Azido-L-Phenylalanine is very important for good expression. During my experiment, I measured the chemical right amount but I was not able to dissolve all the chemical in water even after 3h rotation. So I just added semi dissolved chemical into the bacterial culture for expression. Can anyone suggest me what is the best way to get a high expression of my protein or best way to dissolve the chemical.
The solubility of phenylalanine increases dramatically at pH values above 10 or below 2. You could try dissolving the compound in 0.1M NaOH or 0.1M HCl, as long as your cell culture medium is properly buffered, before addition to the bacterial culture.
Thanks Colin for your suggestion, I will check the buffering activity of culture media with 0.1M NaOH or 0.1M HCl to get an idea to dissolve my chemical in.
Hi there,
According to santa cruz biotech, it is soluble at 5% in 80% AcOH as a solvant.
In this paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2866270/
the solution is prepared as follow : "4-Azido-l-phenylalanine (Bachem) was dissolved in 0.2N HCl and then neutralized with 10N KOH, to prepare a solution with a final concentration of 10 mM".
What is actually the concentration required in your cell culture?
We used small volume of 1N NaOH to dissolve p-azido-Phe and add it to the autoclaved LB. It works well in terms of pAzF incorporation into the target protein. See our published protocol for details.
Analysis of protein ligand-receptor binding by photoaffinity cross-linking.
Wu L, Xu B.
Curr Protoc Protein Sci. 2015 Feb 2;79:19.26.1-14. doi: 10.1002/0471140864.ps1926s79.
@ Dominique liger - the final concentration we are planning to use is 5mM. Thanks for the reference.
@Bin Xu - Thanks for the reference.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression