Troubleshooting Guide for Affinity Chromatography of Tagged Proteins

The troubleshooting guide below addresses problems common to the majority of purification products discussed in this chapter, as well as problems specific to a particular method. In the latter case, the relevant product is indicated.

For more product-specific troubleshooting, refer to the instructions supplied with the product.

Problem	Possible Cause	Solution
Column or plate wells have clogged. OR Liquid has not been completely removed during centrifugation (SpinTrap columns). OR Flow rate is too slow (GraviTrap columns).	Cell debris is present.	Optimize sample pretreatment before the next sample loading.Centrifuge and/or pass the sample through a 0.22 or 0.45 µM filter. Clean the chromatography medium according to Appendix 1 (Characteristics of Ni Sepharose, Ni Sepharose excel, TALON Superflow, and uncharged IMAC Sepharose products). If cleaning-in-place (CIP) is unsuccessful, replace the medium/prepacked column. Try using a HisTrap FF crude column or a HiTrap TALON crude column.
	The sample is too viscous due to too high a concentration of material or the presence of large nucleic acid molecules (may be evidenced by increased back pressure).	Increase the efficiency of the mechanical cell disruption (e.g., increase sonication time). Keep the sample on ice to avoid frothing and overheating because this may denature the target protein.Increase dilution of the cell paste before lysis, or dilute after lysis to reduce viscosity.If the lysate is very viscous due to a high concentration of host nucleic acid, continue cell disruption until the viscosity is reduced, and/or add an additional dose of DNase and Mg2+ (DNase I to 5 µg/mL, Mg2+ to 1 mM), and incubate on ice for 10 to 15 min. Alternatively, draw the lysate through a syringe needle several times.Freeze/thaw of the unclarified lysate may increase precipitation and aggregation. Mechanical lysis of the thawed lysate can prevent increased back-pressure problems when loading the column.If the purification has been performed at 4 °C, move to room temperature if possible (sample viscosity is reduced at room temperature).Decrease the flow rate during sample loading.
	Protein is difficult to dissolve or precipitates during purification.	First, screen for suitable conditions for solubility; vary pH, ionic strength, protein concentration, detergent, or other additives that may affect solubility of the protein.The following additives may be used: up to 1%NP-40, 1% CHAPS, 1.0 M NaCl, 20% glycerol, 10 mM β-mercaptoethanol, 1 to 3 mM DTT or DTE (up to5 mM is possible but depends on the sample and the sample volume). Mix gently for 30 min to aid solubilization of the tagged protein Note that NP-40 (but not Tween™-20 up to 1% concentration) have a high absorbance at 280 nm. Furthermore, detergents cannot be easily removed by buffer exchange.Inclusion bodies: the protein can usually be solubilized (and unfolded using common denaturants such as 4 to 6 M Gua-HCl, 4 to 8 M urea, or strong detergents. See Chapter 10, Handling of inclusion bodies.
There is no or low yield of histidine-tagged protein in the purified fractions.	No histidine-tagged protein present in the starting material.	Verify presence of histidine-tagged protein in the starting material, e.g., by Western blotting.
	Nickel ions are being stripped.	Change to Ni Sepharose excel if the sample causes stripping, for example, if the histidine-tagged proteins are secreted into eukaryotic cell culture supernatants.Ni Sepharose excel, HisTrap excel, and His Mag Sepharose excel have exceptionally strongly bound nickel ions.
	Elution conditions are too mild (histidine-tagged protein still bound).	Elute with increasing imidazole concentration or decreasing pH.
	Protein has precipitated in the column or wells.	Decrease amount of sample, or decrease protein concentration by eluting with linear imidazole gradient instead of imidazole steps.Try detergents or change NaCl concentration.
	Nonspecific hydrophobic or other interactions are occurring.	Add a nonionic detergent to the elution buffer (e.g., 0.2% Tween-20) or change the NaCl concentration.
	Low yield of eluted protein when loading a highly unclarified sample.	Clarify the sample before loading on columns or in wells. High levels of host proteins and other particles may interfere with the binding of the target protein. Consider using a HisTrap FF crude column or a HiTrap TALON crude column.
	Histidine-tagged protein is not completely eluted.	Elute with a larger volume of elution buffer and/or increase the concentration of imidazole.
Histidine-tagged protein is eluted during sample loading/wash.	The concentration of imidazole in the sample and/or binding buffer is too high.	Lower the imidazole concentration. Note that imidazole is usually not required in sample and binding buffer for TALON Superflow and Ni Sepharose excel.
	The histidine tag may be insufficiently exposed.	Perform purification of unfolded protein in urea or Gua-HCl as for inclusion bodies to investigate if the tag is hidden. To minimize dilution of the sample, solid urea or Gua-HCl can be added. Consider a His-10-tag or add a linker between the tag and the target protein to increase exposure.
	Buffer/sample composition is not optimal.	Check pH and composition of sample and binding buffer. Ensure that chelating or strong reducing agents are not present in the sample at too high concentration.Increase the buffering capacity of the wash and elution buffer to avoid a drop in pH caused by imidazole.
	Histidine tag has been lost.	Check sequence of the construct on a Western blot or extract using anti-His antibody.
	Incubation time is too short.	Decrease the flow rate or increase the incubation time of the sample in the wells/batch or use a lower centrifugation speed/vacuum (SpinTrap and MultiTrap columns).
	Contaminants may have a high affinity for certain metal ions.	For applicable formats (i.e., prepacked HisTrap columns), join two or three columns together or change to a larger column.
	Capacity is exceeded.	For applicable formats (i.e., prepacked HisTrap columns), join two or three columns together or change to a larger column.
Histidine-tagged protein is found in the pellet.	Sonication may be insufficient.	Cell disruption may be checked by microscopic examination or monitored by measuring the release of nucleic acids at A260. Addition of lysozyme (upto 0.1 volume of a 10 mg/mL lysozyme solution in 25 mM Tris-HCl, pH 8.0) prior to sonication may improve results. Avoid frothing and overheating because this may denature the target protein.
	Protein was adsorbed to cell debris during extraction and lost upon clarification.	Change extraction conditions (pH, ionic strength, try detergent solubilization).
	The protein may be insoluble (inclusion bodies).	The protein can usually be solubilized (and unfolded) from inclusion bodies using common denaturants such as 4 to 6 M Gua-HCl, 4 to 8 M urea, or strong detergents. See Chapter 10, Handling inclusion bodies.
The eluted protein is not pure (multiple bands on SDS-PAGE).	Proteases have partially degraded the tagged protein.	Add protease inhibitors (use EDTA with caution).
	In vivo anomalies of protein biosynthesis, e.g., premature termination of translation.	Change fermentation and induction conditions. Consider changing host strain or host to overcome problems with codon bias.
	Contaminants have high affinity for the metal ion.	Elute with a stepwise or linear imidazole gradient.Wash before elution with binding buffer containing as high a concentration of imidazole as possible, without causing elution of the tagged protein.Try TALON Superflow for high purity or test using another metal ion on IMAC Sepharose.A second chromatography step such as SEC may be necessary.
	Contaminants are associated with tagged protein, e.g., chaperone attached to the target protein.	Add detergent and/or reducing agents before sonicating cells. Increase detergent levels (e.g., up to 2% Tween 20), or add glycerol (up to 20%) to the wash buffer to disrupt nonspecific interactions. Increase the imidazole concentration or change the metal ion used for purification.Increase the sodium chloride concentration up to 500 mM in buffers.
	Unbound material has been insufficiently removed by the washing step.	Repeat the wash step after sample application to obtain optimal yield and purity.
Unwanted air bubbles have formed when using a chromatography system.	Unclarified lysates may cause increased air bubble formation during purification.	An attached flow restrictor in the chromatography system after the column and detector flow cells can prevent this. If a flow restrictor is attached, it is important to change the pressure limit to 0.5 MPa (5 bar) on the ÄKTA system (where the column and the flow restrictor give a pressure of 0.3 MPa and 0.2 MPa, respectively).
MultiTrap: Leakage of solution is observed after removing foils.		Add 500 µL of deionized water twice before adding binding buffer to the wells. Remove the solution between the additions with either centrifugation or vacuum.

Materials

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