I have a problem for several months. I desperately need your help.
I have two tagged proteins GST-A and His-B, both of them are RNA binding protein, pI of GST-A is 6, pI of His-B is 9. Previous reports says A and B has interaction, but in the presence of RNA, the interaction will be disrupted.
My aim is to purify this A-B complex, and try to determine the crystal structure.
During my purification, after sonication, I treated with 0.2 mg/mL RNase A in 35℃ for 30 min. Then the tagged A and B protein were purified by Ni-NTA or GST beads with buffer containg 1.5 M NaCl, after removing the tags, desalting to 100 mM NaCl, mix in ratio, and load on gel filtration/size exclusion chromatography with 100 mM NaCl, there is completely no binding between A and B, and I found there is still RNA in the peak based on UV 280/260.
If so, how can I completely remove RNA? Thanks a lot.
It could actually be RNA or DNA given a peak absorbance at 260 nm, so treating with a combined RNase/DNase may be beneficial (such as Benzonase).
Adding another step to your purification, such as Ion exchange, also helps to remove contaminating nucleic acid. Alternatively you could try on column protein refolding (link attached for an example). This can work well for His-tagged proteins but the GST fusion could be problematic.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2624574/
It could actually be RNA or DNA given a peak absorbance at 260 nm, so treating with a combined RNase/DNase may be beneficial (such as Benzonase).
Adding another step to your purification, such as Ion exchange, also helps to remove contaminating nucleic acid. Alternatively you could try on column protein refolding (link attached for an example). This can work well for His-tagged proteins but the GST fusion could be problematic.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2624574/
Hi there,
Purification on Heparin-Sepharose is usual for removing bound nucleic acid from nucleic acid binding proteins. Heparin acting as a competitor towards nucleic acid for the binding to proteins, this technique is comparible to affinity chromatograph.
Hi Zixun Han,
As Gavin suggested, you can use a combination of DNase and RNase to get rid of any nucleic acid contamination. Also, you can increase the dose of RNase A as well the incubation time.
Good Luck!
Utkarsh
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