ZAGENO

Protein Purification Troubleshooting

Introduction

Protein purification can be a bit tricky. While specific kits targeted towards purifying protein make such experiments part of a standard routine, there are myriads of guides and alternatives that might apply for each method. So there is always the possibility for one or more steps to go wrong or cause problems in experiments with this level of complexity.

If you are experiencing an issue, don't worry! ZAGENO's troubleshooting guide on protein purification is here to help.

free shipping all brands

Protein-Column Binding Does Not Occur

  • Binding could not occur due to insufficient time: If a recombinant protein was expressed in an inducible system, check that the induction worked properly
  • Protein tag is inaccessible for binding or has not been properly translated: Allow for binding by moving the position of the tag. Ensure plasmid sequence is correct
  • Unsuitable binding conditions: Adjust the characteristics of your buffer, (pH, concentration)

No Protein in the Eluate

  • The protein is not expressed: If a recombinant protein was expressed in an inducible system, check that the induction worked properly
  • Protein quantity below the protein binding threshold: If you are using an affinity column, try using a higher input amount
  • High affinity between tag and ligand: Adjust the characteristics of your buffer, (pH, concentration) or increase competitor concentration
  • Protein still bound to column: Prepare a fresh elution solution and repeat your elution
  • Protein aggregated on column: Adjust the characteristics of your buffer, (pH, concentration)

Low Resolution

  • Protein aggregated on column: Adjust the characteristics of your buffer, (pH, concentration)
  • Flow rate is quicker or slower than the optimum: Alter the rate of flow until the optimum is achieved
  • Column was insufficiently washed: Use a more stringent buffer
  • Elution conditions lack stringency: Adjust the characteristics of your buffer, (pH, concentration) or increase competitor concentration

Target Protein Elutes with the Washings

  • Washing too stringent: Reduce the number of washings or optimize your washing buffer
  • Saturated column or resin: The column is saturated and cannot bind to any more protein. Lower the protein concentration in your input

High Background / Elution of Contaminants

  • Washings are not stringent enough: Include additional washing steps or optimize your washing buffer composition
  • Wrong buffer: Consider additional purification steps or lower the total amount of protein loaded on your column
  • Resin bind to specific proteins: Consider additional purification steps or lower the total amount of protein loaded on your column

Protein Degradation

  • The protein was degraded in the starting material Flash freeze and store your samples at -80 °C
  • Sample processed at high temperatures Grind your samples in liquid nitrogen and process them on ice or in a cold room at 4 °C

If you plan on repeating this experiment, follow these tips to help minimize mistakes

  • Store all your samples for protein extraction at – 80 °C
  • Process your samples on ice or at 4 °C
  • Use five volumes of lysis buffer for every gram of cells
  • Always use Imidazole in the elution buffer for His-tagged proteins
  • Save aliquots for all the washing steps for diagnostics
  • Run all your samples, including washing step samples, on a gel to follow your protein of interest during your purification. Use silver staining for the gel or western blot, if you have a suitable antibody
  • If you are using an inducible expression system for your protein, check that the protein is expressed using an SDS page before starting the extraction

Support

Hopefully by following these protein purification tips, your problems will soon be a thing of the past. If you still have issues - or have extra tips - let us or the community know in the comments.

Discussion