Dear all,
Anyone can suggest me regarding a precipitation issue in elution of Anion Exchange chromatography, After elution thread like particles are forming in elution pool. We are using PB buffer with NaCl and presence of EDTA. My protein is carrier molecule having PI value 5.8.
if you do not mind salt concentration, you may add some higher salt buffer in the collection tubes before you elution, allowing the eluting protein directly go to higher salt buffer.
Not all proteins are happy in high salt concentration: on an ion-exchange you elute the protein around 200/300mM NaCl and what you are experiencing most likely is salting out. You could try running a desalting column right after the ion exchange or put your protein in dialysis after elution. One final possibility could be the presence of EDTA and your protein being a Ca++ or Zn++ binding protein.
Dear Both,
Thanks for the valuable information and kind reply, exactly you both are right, by increasing buffer strength we can reduce the precipitation issue but molecule stability may be issue, i will try in next experiment.
In my process of IEC i am eluting at 10mM PB and 75mM Nacl with 0.25mM EDTA, buffer strength 10mS, pH 7.5. I tried with buffer change, increasing buffer strength. Till problem not get solve.
Hi Nagabhushanam, are the thread-like particles you observe after the anion exchange your protein or some other stuff?
Improper storage of samples can be a good medium for fungi to grow, or it could be DNA/RNA forming these strands.
If these strands are your protein of interest, then they are probably aggregating through hydrophobic interactions. You could include 0.1% TX-100 in your purification procedure and see if that improves the solubility.
Hi thank you Stefansson for your suggestion, those particles are exactly protein of interest only. We checked 0.8% agarose gel but did not see any nucleic acid bands. By adding detergent if it get soluble then problem will solve. I have to do dialysis to remove detergent. I will try from 0.01% on wards.
If this is an anion exchange what is your binding pH? You can use 200-300mM salt to elute but also shift the pH of the elution buffer. Adding a small amount of non ionic surfactant can also help keep the protein in solution. If you provide more information regarding your binding and elution conditions, I could provide additional feedback.
Remove detergent with a dialysis is not an easy task, especially Triton X-100. In order to help you better is need that you tell us more about your purification protocol. Which is the source of your protein? How do you prepare your sample before load to the column? As Kou Hayakawa said, is better to improve the environment to maintain the protein in better conditions.
I can suggest you to use a small concentration (0.01%) of a non-ionic detergent as dodecyl maltoside, this detergent had shown to maintain the activity of a lot of membrane protein and can be useful. Also you can add some glycerol to your buffers to avoid aggregation as well as cryo-protection if you want to store your sample at -80 °C.
Dear Laurie Broadwater thanks for your suggestion. These are conditions i am using right now.
Equilibration,Binding,Washing pH is 7.5 ( Buffer composition is 10mM PB+10mM NaCl+0.25mM EDTA) and Conductivity is 3.5 mS/cm. Elution pH 7.5 and conductivity is 10.5mS/cm (Buffer composition is 10mM PB+75mM NaCl+0.25mM EDTA).
I am waiting for your reply.
Thank you.
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