Troubleshooting Guide for Purification using NEBExpress™ Ni Spin Columns

Problem

Possible Causes

Solution

Low protein yield

or

No protein eluted

Low expression of target protein

  • Check the expression level of the protein in the crude extract by running an SDS-PAGE protein gel.
  • Load a larger quantity of crude extract onto the column.
  • Concentrate the crude extract prior to loading on the column.

Target protein did not bind to the nickel resin

  • If you’ve prepared your own lysis/binding buffer, ensure the pH is above 6.5 and the buffer does not contain imidazole.
  • Perform a western blot to determine if a His-tag is present. If the blot is positive, it is possible that the tag is sterically blocked from binding to the resin; attempt purification under denaturing conditions, change location of His-tag (N vs C terminus) or add linker between His-tag and target protein in fusion construct.

Target protein is still bound to the resin

  • Confirm that target protein is bound to the resin by taking an aliquot of the resin, stripping with SDS gel loading buffer and analyzing via SDS-PAGE.
  • Ensure the Elution Buffer was correctly prepared to contain 500 mM Imidazole.
  • Mix the column resin with Elution
  • Buffer end over end at 4°C for 30 minutes to 1 hour before centrifuging.

Target protein is degraded, or the his-tag is missing

  • Include protease inhibitors in the Lysis/Binding Buffer.
  • Perform a western blot to determine if a His-tag is present.
  • Consider making a new construct or choosing a different E. Coli expression strain.

Protein eluate is not pure

Number of washes is not sufficient

  • Increase the number of washes.
  • Make sure the resin is thoroughly mixed with the wash buffer prior to centrifuging.

Concentration of imidazole is too low

  • Increase the concentration of Imidazole in the wash buffer up to 15mM.

Second eluate contains more target protein than the first eluate

Resin not mixed uniformly with the elution buffer

  • Ensure thorough mixing of the column resin with elution buffer.
  • Extend end over end mixing at 4°C for 30 minutes to 1 hour before centrifuging.