Protein samples may contain solid impurities. Mostly, it’s because bacterial lysates are loaded directly. It is recommended to process protein by microfiltration.

· His tag is not present, which is possibly due to incorrect vector construction, premature termination of expression (His tag on C-terminal), the internal ribosome entry site (His tag on N-terminal), etc. It is recommended to sequence or change the expression vector.

· His Tag is inaccessible folded and buried inside the protein's three-dimensional conformation, which happens mostly when denatured inclusion bodies are purified. It is recommended to fully denature protein by adding stronger denaturants (Use 6 M Guanidine hydrochloride to replace 8 M urea), Cosolvents (SDS) or reducing agents (DTT orβ- Mercaptoethanol).

·  Buffer composition is incorrect. When Ni-NTA resin or Ni-IDA resin were used to purify His tag protein, some components in the sample buffer and equilibration buffer may interfere with the binding of protein.

Some recommended concentrations:

EDTA: ≤0.1 mM (Not Recommended to use);

DTT: ≤1 mM (Not Recommended to use);

β- Mercaptoethanol: ≤20 mM (Use cautiously);

Imidazole: ≤20 mM (Protein dependent).

·  Elution conditions are not optimal. It is recommended to apply Imidazole or pH gradient elutions. The optimal elution condition varies from protein to protein, which needs to be optimized step by step.

·  Contaminant proteins non-specifically bind to the purification resin. Add Imidazole (The recommended concentration is ≤ 20 mM which depends on protein) to sample buffers and equilibration buffers to inhibit non-specific bindings.

·  Contaminant proteins non-specifically bind to the target protein. Add non-Ionic detergents (Triton X-100: ≤1%), Glycerol (≤10%), NaCl (≥300 mM), reducing agents (β- Mercaptoethanol: ≤20 mM) or some other components to sample buffers and equilibration buffers.

·  Protein has been degraded. It is recommended to purify protein at low temperature and add protease inhibitors (such as PMSF) into sample buffers and equilibration buffers.

·  Premature termination of expression (His tag on C-terminal) or internal ribosome entry site (His tag on N-terminal) is present. It is recommended to change the expression vector.

·  Protein content is too low. It is recommended to replace Coomassie Blue Staining with detection methods with higher sensitivity, such as Silver Staining and Western Blot. Alternatively, optimize upstream conditions of protein expression to obtain more protein.

·  Protein does not bind to the resin. It is recommended to detect protein content of the sample, the flow-through fraction and the wash fraction.

·  Protein binds to the resin too strongly. It is recommended to enhance elution strength using higher concentration of Imidazole or lower pH.