I am trying to purify a heterologous protein with His tag in yeast but I am facing issues with binding my protein to Ni-NTA beads. Please suggest some methods to purify my protein of interest.
These are the conditions I have followed
My protein of interest was precipitated from Pichia X-33 cells culture supernatant (400 ml) on day 4 using 60% of ammonium sulphate. The precipitated protein pellet was dissolved in 6 ml of buffer (Tris-20 mM, NaCl-500 mM, pH-8). 6ml of dissolved protein was dialyzed in 3 cycles of dialysis (Buffer: Tris-20 mM, NaCl-500 mM, pH-8). Dialyzed protein was purified using Ni-NTA agarose beads. My protein PI:6. I have used the same dialysis buffer with 20 mM and 40 mM imidazole for washing.100 and 300 mM imidazole for elution. My protein is not binding to Ni-NTA agarose beads. I have tried at pH:7.4 also. In all the cases it's in flowthrough.
I have attached the gel image also.
.
Hi there,
You should try IEC with an anion exchanger (DEAE for instance) as according the info your protein should be negatively charged at pH8. BTW 5mM NaCl sound very low...
I tried both cation and anion exchangers but still, my protein din binds to the column.
Sorry, it's 500mM Nacl and not 5mM Nacl.
Thanks for the kind reply
Hi again, to run IEC you should work at low salt concentration at the binding step (salt ion is a competitor to bind the matrix). I would suggest not more than 50mM for an efficient binding.
Thank you, Dominique Liger Itabajara Vaz Jr.
I will try low salt concentration.
Hi,
Adding 20-30 mM Imidazole in your lysis and dialysis buffer may help in binding the protein to Ni-NTA.
Check the beads efficiency using control sample and regenerate once if still you are facing the issue.
1. Increase the concentration of the Ni-NTA beads. 2. Make sure the pH of the lysis buffer is optimal for the His tag. 3. Increase the salt concentration of the lysis buffer, as this can help the protein bind to the beads. 4. Optimize the lysis buffer to include reducing agents such as Tris-HCl, DTT, and β-mercaptoethanol, as these can help to stabilize the His tag and aid in binding. 5. Increase the time for incubation of the lysate with the Ni-NTA beads, as this may increase the chances of your protein binding to the beads. 6. Make sure the protein is not degraded by proteases during the lysis procedure. 7. Increase the temperature of the lysis buffer to 37°C, as this may help to increase the binding affinity of the protein to the beads. 8. Try using a different Ni-NTA resin, such as IMAC resin, as this may be more efficient at binding your protein. 9. Try using a different His tag, such as a polyhistidine tag, as this may be more efficient at binding the protein to the
- Badges
- Science topic