I have purified my protein which MW is 27kDa for crystallization purpose but some impurities are there. So I want to do HPLC for further purification but I am not having exposure for HPLC work, I am confuse how to decide solvent system to my protein for HPLC. what parameters I should keep in mind before HPLC run and how to select solvent system.
When the denaturation of your protein does not play a role (which it might), I would recommend a C3 column and a 0.2% formic acid and acetonitrile gradient. Works for most proteins. If you have a very hydrophobic or sticky protein, than you may need to develop a specific method for your particular protein using different solvent systems.
If denaturation of your protein plays a role, HPLC may not be your best option since many solvent systems would denature your proteins. Exceptions may be some water based solvent systems that you use in conjunction with size exclusion columns.
A final note, the size of your column really depends on the amount of protein that you have.
Hope this helps.
I would recommend a liner gradient of 0.1% tri fluro acetic acid and acetonitrile.
Well, you don't need to do HPLC for final polishing of your protein for crystallization. Rather, go for AKTA- FPLC systems. As you quoted that your protein is 27 kDa, I would recommend S75 column from GE healthcare. Regarding the buffer: you need a low concentration buffer like 20mM Tris or any other depending on the pI of your protein, also keep 150-200 mM NaCl and some reducing agents depending on the availability of free cysteines in your protein.
Good luck with your experiments!
One can buy the same types of columns for HPLC that are used for FPLC. The HPLC columns just have better resolution. So what one uses depends on the impurities present. If the impurities are either higher or lower MW than your protein, then a HPLC sizing column could be used with buffers of any kind that are water based. One would only use the HPLC column if major impurities come out closely using conventional FPLC do that better resolution is needed. I would be afraid to use anything that might denature your protein, like acids and organic solvents, right before crystallization unless you know the acids and solvents will not denature your protein.
For ion exchange, and other columns, these could be used as well if you want to get better resolution. Again, the buffers used with the FPLC are the same for HPLC.
If acids and solvents are required, then you can test your protein by CD and other methods first to make sure that it will survive the column conditions.
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