What should I do if purified protein fails to elute from TALON resin?
What should I do if purified protein fails to elute from TALON resin?
Check to see if any of the following factors are responsible and try the recommended solutions listed below:
1. Is the protein being expressed?
Check the lysate using Western blotting, preferably with a 6xhis antibody. This also assures that the reading frame is the same for the protein and the tag.
2. How does the lysate preparation look?
Was the lysate preparation turbid? If not, the cells may not be lysed completely. Try adding more lysis buffer, perform freezing/thawing of the sample, and incubate with the lysis buffer for a longer period of time.
Additional recommendations for disruption of membranes: Sonication is quick, but can be tedious if there are multiple samples. Alternately, up to 1% nonionic detergent, e.g., Triton, NP-40, or Tween can be used (SDS, CHAPS, and sarkosyl may cause problems).
3. Is the protein binding to the resin?
Check the flowthrough and wash fractions via Western blotting to see if the protein is bound (and not eluting), or flowing through during the washing steps instead of the eluting steps (i.e., not binding in the first place). If the protein does not seem to be binding and is flowing through:
*Determine whether pH- or imidazole-based buffers are being used for elution.
*Check buffer compositions for incompatible reagents.
*Check the imidazole concentration; concentrations above 20 mM can inhibit binding.
*Check the buffer pH; pH values below 6.8 can inhibit binding.
*Check for the presence of supplements in the cell culture medium.
*Cysteine, tryptophan, histidine, or other charged amino acids can prevent his-tag binding.
*Check the solubility of the protein:
+Treat a small aliquot (1 ml) of lysate with 6 M guanidine.
+Apply the protein to 50 µl of TALON resin.
+Perform a mini-scale purification.
*Try adding a nonionic detergent to improve solubility.
*Shearing DNA or adding DNase I decreases viscosity and improves protein binding.
4. Is the protein eluting from the resin?
*Check the flowthrough and wash fractions via Western blotting to see if the protein is bound (and not eluting), or flowing through during the wash steps instead of the elution steps (i.e., not binding in the first place).
*Alternatively, boil a small quantity of the beads in gel loading buffer and analyze the supernatant on a gel after boiling to confirm that the protein is not eluting. These methods should make the protein come off the resin; however, the resin, and probably the protein, will not be reusable.
*If the protein does seem to bind but does not elute:
+Determine whether pH or imidazole-based buffers are being used for elution.
+Check buffer compositions—imidazole concentrations may need to be increased to allow elution.
+Check the buffer pH—it may need to be lowered slightly to allow elution.
*If no protein band is seen after stripping the resin with EDTA, the protein may have bound nonspecifically to the resin. If the protein is actually bound to the Sepharose and not the metal, the only method for removing the protein is to boil it for 10 minutes with 1% SDS. Adding the detergent can facilitate removal of the protein. This problem has been known to occur with lectins.
5. Is the protein eluting with other contaminating proteins?
*Determine whether pH or imidazole-based buffers are being used for elution.
*Try increasing the stringency of the wash.
*Check buffer compositions; the imidazole concentration may need to be increased to improve purity.
*Check the buffer pH; it may need to be lowered to improve purity.
*Try increasing the salt (counterion) concentration (NaCl or KCl) to 0.5 M.