Hello. Hope everyone is doing well. i purified my protein with the Q column (ion exchange column). The first time everything was ok but when I tried for the second-third time i got lots of DNA contamination along my target protein. My protein includes no tag and also tried to change the protein buffer from PBS to PBS(2M NaCl) using a gel filtration column and again change to normal PBS. I lost half of my protein and the left one also was the same. please let me know if you have any suggestions. thanks
Hello,
When purifying proteins, take the following actions to reduce or remove DNA contamination:
1. Ensure that all equipment and chemicals are clean. To avoid cross-contamination, use equipment specifically designed for handling and purifying DNA. Use water and reagents that have been shown to be DNA-free.
2. Decontaminate: Make all of the equipment, including chromatography media, purification columns, and other surfaces clean using a DNA-specific decontamination solution.
3. Degrade any remaining DNA or RNA contamination by treating pure protein samples with a nuclease enzyme, making sure that it is as active and specific as possible.
4. Alternate buffers: Think about switching out the PBS (2M NaCl) or Tris-based buffers that are utilized in the purification technique.
5. Optimally configure the purification process: boost selectivity by modifying buffer composition, pH, or salt concentration. If you want to effectively extract nucleic acids, think about using alternate purification methods such affinity chromatography or size-exclusion chromatography.
6. Carefully handle and store the purified protein sample using sterile procedures, specialised tubes, and gloves. Store at the proper temperatures and humidity levels.
7. Carry out quality control inspections: To minimize or completely eradicate DNA contamination, regularly verify the purity of proteins, do tests for DNA contamination, and constantly watch the purifying procedure.
Regards
Hello,
Gayatri Munieswaran answer 4 is very relevant :
4. Alternate buffers: Think about switching out the PBS (2M NaCl) or Tris-based buffers that are utilized in the purification technique.
Indeed i found that some DNases (DNase1, benzonase ...) were inactive in phosphate buffers.
So, if you choose to remove DNA enzymatically, avoid using a phosphate buffer, like PBS.
Otherwise you can try to break the DNA mechanically by, passing your sample through syringe needles or by sonicating it or ....
Regard
Thanks everybody for the responses. Dear David you think if I pass my sample through the o.22uM syringe filter it can work for me? My elution buffer is not PBS. Its 50mM sodium acetate +1M NaCl.pH.5.5. I just used PBS as I saw this in one protocol for removing DNA. I purified and used Gel filtration column to change to high salt PBS and again changed to 1× PBS and nothing happened except losing lots of my antigen.
Dear Gayatri, really thankful for spending time and suggesting so many tips. To be honest the equipments I use are not specific so I cannot take care of them and avoiding contamination. As so many other students use these equipments and no problem with them then I think it cannot be related to the devices. After centrifuging I check the medium contains my protein and it contains lots of DNA cintaminant and the ddH2O I used before and no problem. My elution buffer is not PBS. After using Ion exchange column to purify, I used gel column to change the buffer to PBS. One of your suggestion took my attention. Can I use DNase enzyme to digest the DNA? maybe this method worlds for me. I purify protein using ion could and use DNase to remove DNA?
REGARDS
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