I need to dialyze my protein to remove NaCl (300 mM) and imidazole (the current buffer also has 10 mM Tris HCl, pH 7.4). I want to exchange the protein to a buffer that only contain 10 mM Tris HCl, pH 7.4, but I am concerned about the possibility of precipitation due the lack of NaCl. Could you give any suggestions?.
See section 2.2 in this paper.
https://www.uic.edu/orgs/ctrstbio/manuals/kelly.pdf
See section 2.2 in this paper.
https://www.uic.edu/orgs/ctrstbio/manuals/kelly.pdf
This is my favorite from the same autors:
https://www.uic.edu/orgs/ctrstbio/manuals/kellybba.pdf
See Table 1
Hi Carmen, that's strongly depends of what kind of experiment you wanna do, I recommend you the following papers
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2752288/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2728378/
http://link.springer.com/protocol/10.1385/1-59259-762-9%3A055
Hope this help you!
If you are interested in going to well under 200nm you will have to find replacements for Tris, imidazole, and NaCl. I realise this is not always so easy. If the goal is to go to 180nm, weak phosphate buffer with NaF is often helpful. High protein concentrations and cuvettes with the shortest pathlength possible will minimise buffer background. You will need to keep total absorbance well under 1.
Good luck.
I am using CD and have several questions about the pH of its designated buffers.
1. What is the pH per se of a freshly prepared 10 mM NaH2PO4 water solution ?
2. What is the pH per se of a freshly prepared 10 mM NaH2PO4 , 150 NaF water solution
3. What is the pH per se of the above solutions when Na is replaced by K.
5. What is the pH per se of a 50 mM Tris (no HCl) water solution.
4. Does the phosphate has enough ''strength ' to keep the pH constant over time, say 1-2 weeks ?
5. What is the UV cutoff of a 50 mM Tris (no HCl) water solution.
Thank you.
Valentin
For phosphate (and other common) buffers, there are tables and calculation tools, such as these ones:
http://www.uslims.uthscsa.edu/po4buffers.php
https://www.liverpool.ac.uk/pfg/Research/Tools/BuffferCalc/Buffer.html
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