I am facing problem during purification of cytosolic fusion protein, by using Qiagen kit spin column (binding capacity 300 ug of protein)
80% protein is coming out in flow through so less yield is reduced during elution.
I am trying to fix it but still not successful how to troubleshoot this?
Dear Monika,
Did you check the suggestions in the Troubleshooting section of the handbook that comes with the column? There are many possible reasons for low binding efficiency and the data you provided is just not sufficient to give a very good suggestion about what to improve. It could be that the binding conditions do not fit or the tag was split off, to mention just two of the possible reasons.
1. Reduce the concentration of imidazole in the adsorption buffer, if any
2. Increase the contact time between the sample and the adsorbent
3. Reduce the presence of small competing molecules, if any
4. Use western blot to check for the presence of the his-tag
5. Adsorb the protein under denaturing conditions to determine if the tag is hidden
6. Switch to Cu ions
Hi there,
How much protein do you load onto the column and how much do you actually recover with elution? It might be that you simply saturate the matrix. An other obvious possibleissue would be the actual presence/accessibility of the tag as mentioned earlier.
Hello Liger, I loaded only 270ug & binding capacity of column is 300ug so I recovered with 80ug of protein in elution. His tag is in frame I cross checked it with sequencing result of clone. The column which I am using is mini column contains Ni NTA resin. Will Ni NTA beads help for this purification ?
Hello Dolinar, yes I have gone through with troubleshooting section for Qiagen kit mini spin column still not getting expected recovery in elution. You mentioned about bind condition so how to improve that?
Tag is in frame as confirmed by sequencing of recombinant clone before expression of protein.
The theoretical binding capacity of 300 ug is obtained under equilibrium conditions. This means that it occurs for an infinite contact time between protein and nickel ions. 80 ug is not that bad.
Hello Omar, following answers for N- Nus & C- His tag protein .
1. I tried point 1 & 2 still no luck (adsorption buffer means equilibration buffer?).
3. There is no competing molecules as purification buffers contain Phosphate buffer , Nacl & imidazole.
4. I confirmed the presence of His tag by sequencing of recombinant clone before starting expression. Now is that necessary to check it by western blot ?
5.Kindly clear this point adsorb protein under denaturing condition? Here protein should be in denaturing condition or should I follow the denaturing condition purification (As buffers are different for Native & denaturing purification)?
I tried only native purification protocol
I did not try denaturing condition with N- Nus tag & C- His tag protein as I am getting soluble protein after expression (Native purification protocol) but when same protein was ONLY with C- His tag that time after expression it formed inclusion body which I tried to dissolve by Denaturing conditions (Successful) after expression clear & intense band observed but after purification (Native purification) in that same denaturing condition very faint or NO band is observed during elution irrespective of dialysis.
6 Is Cu ions capable for N- Nus tag & C- His tag protein purification ?
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