I have GST and HIS tag purified recombinant proteins and would like to go for CD analysis. Does someone have the sample preparation protocol for running CD analysis? I would also like to know the appropriate concentration of my purified protein for good result.
To make things easier I am attaching few articles which should help you answer those questions. I tried to attach couple of more but I couldn't. Good luck
There are plenty of papers talking about CD which helped me to plan my expt. long time ago. good luck
The imidazole from His tag elution will cause a severe problem in the CD spectrum because it absorbs at the same wavelength as the protein (the same applies to glycerol, DMSO, DTT, some detergents, and most other buffers besides Tris and phosphate). You will probably need to dialyze the protein against another buffer, preferably phosphate buffer with NaF substituted for NaCl.
Jeffrey is right. While NaCl does not give you a CD signal but it is absorbs uv light very efficiently below 200nm. On thing to do is to watch the PW voltage. If that is >700 you do not get a useable signal anymore. It is possible to have NaCl etc present, but in order for them not to interfere the concentration must be small. You could for example use a CD cell that is 0.01 to 0.1mm thick. Of course now you have to have your protein at high concentration which may have problems on its own. Just watch the PM voltage.
Dear All,
I am very happy to have yours valuable opinions. I will quickly follow all those suggestions while preparing protein sample for CD analysis.
To add to the answers above.
Prepare the protein at 0.5 mg/mL maximum (for a 0.02 mm cuvette) in the simplest buffer possible i.e. avoiding multiple components and ions. In our hands 50 mM Tris buffer gives good results.
DMSO absorbs strongly in the UV, you will not get a good Cd spectrum. Either remove the DMSO by gel filtration or start over from an ammonium hydroxide solution
You may try making a stock solution of the compound first in acetonitrile then diluting it. Acetonitrile is UV transparent and will probably have little effect on the aggregation at low (1-5% ?) concentrations
Hi all
I have my protein in 20mMTris, 500mMNaCl, pH 7.5, so I dialysed it to change NaCl with 100mMNaF for CD run, BUT my protein got clump and precipitated, please anybody can help??
Thanks
>>I dialysed it to change NaCl with 100mMNaF for CD run, BUT my protein got clump and precipitated
It looks like your protein will not tolerate low salt conditions. The decrease in sensitivity is not that much with 500 mM NaF. Just be sure to use a 500 mM NaF blank
- Badges
- Science method