Protein Purification Support—Troubleshooting
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If the sulfhydryl groups in the peptide are oxidized to form disulfide bonds, the peptide will not bind to the SulfoLink™ resin. Lyophilized peptides are typically supplied in reduced form. The peptide should be dissolved in coupling buffer and immediately coupled to the column. Immobilized TCEP Disulfide Reducing Gel (Cat. No. 77712) can be used to quickly reduce a peptide that has been in solution, with quick recovery of the peptide free of the reducing agent.
Alternatively, TCEP in solution can be added to the peptide to retain it in the reduced form during the immobilization process.
Low pH elution is the most commonly used method of elution for affinity purification, however, it could lead to denaturation of some antibodies which will negatively affect subsequent antigen binding. To prevent this from happening, after protein purification and column regeneration, immediately wash the column with neutralization or binding buffer and store at 4 degrees C in buffer containing an antimicrobial agent such as sodium azide.
Alternatively, use a near-neutral, high salt elution buffer such as Pierce™ Gentle Ag/Ab Elution Buffer (Cat. No. 21013, 21027, or 21030). For alternative elution buffers, please refer to this Tech Tip.
NativePure™ Affinity Purification
- The expression level of your protein may be too low; ensure that the protein of interest is in vivo biotinylated.
- AcTEV™ cleavage is not efficient or the enzyme is inactive; ensure that the AcTEV™ protease is stored at –20 degrees C or –80 degrees C to prevent any loss in activity. You can try increasing incubation time or amount of enzyme to optimize cleavage.
- Protein complex not recovered in the soluble fraction; perform western analysis to check that protein complexes are recovered in the post-nuclear supernatant. If more than 50% of the protein complexes are remaining in the pellet, solubilize the protein complexes using mild, non-ionic detergents, such as NP-40, Triton™ X-100 or equivalent.
- Not enough sample was loaded; increase the amount used.
- Adherent cells are difficult to harvest using mild conditions; if you need to use trypsin treatment, do so for a short time.
- Protein is degraded; perform all purification steps at 4 degrees C. Check to make sure that the BioEase™ tag is not cleaved during processing or purification, and include protease inhibitors during cell lysis.
Complexes can dissociate during lysis preparation or purification. To minimize this, perform cell lysis using freeze-thaw cycles. Avoid trypsinizing cells or scraping cells. Perform cell lysis in the absence of NP-40, as some protein complexes may be unstable in its presence. Avoid vortexing the lysate during preparation, and perform all purification steps at 4 degrees C using chilled buffers.
This typically occurs when protein is overloaded. Load less protein or use more resin for purification.
Try including 0.1% NP-40 during the binding step to eliminate nonspecific binding. Include protease inhibitors during cell lysis to minimize protein degradation.
ProBond™ and Ni-NTA Purification
Unfortunately, if the resin completely froze, then it might not efficiently bind to proteins anymore. A good way to check the quality would be to let the resin thaw. If the resin forms aggregates or clumps, the resin is no longer functional. If you do not see clumps or aggregates, then try testing the resin on a non-precious sample.
Try heating the buffer to 37 degrees C. The precipitate should re-dissolve back into solution. If heating does not help, check the pH. It should be between pH 7.8–8.0, and can be adjusted with HCl or NaOH.
Chromosomal DNA may be the cause. Ensure that genomic DNA is sheared by pulling the lysate up and down a few times through an 18-gauge needle. Ensure further that the lysate is spun for 15 min at 10,000 rpm in a Sorvall™ SS-34 rotor in order to clear the lysate. Filtration of the lysate through a 0.8 μm filter is another option.
The lack of binding could be because the protein already eluted with the wash buffer. This means that the purification conditions were too stringent. To achieve less stringent conditions, try these options:
- Use only 10 mM or less imidazole (1–5 mM) in the binding or wash buffer and /or
- Reduce the NaCl concentration from 500 mM to 250 mM or less; a systematic titration may be necessary, i.e., reduce in increments of 100 mM and then fine tune.
- Try to use an imidazole step gradient.
- The His-tag is hidden due to folding; try a denaturing elution.
The protein may have been eluted because the procedure was too stringent. Reduce the stringency as follows:
- Increase the pH by 0.5 to 1.0 pH unit
- Decrease the NaCl concentration in increments of 50 to 100 mM
Please see our suggestions below:
- Add 0.1% Triton™ X-100 or Tween®-20 to help solubilize further; purify at room temperature if protein is not temperature sensitive. However, keep in mind that most proteins are temperature sensitive.
- If secreted proteins are in media with low pH, they must be dialyzed to avoid Ni2+ reduction.
- If solubility is a real problem (e.g., microsomes), include up to 0.2% Sarkosyl in the 6 M guanidine lysis buffer; this will help to solubilize everything and may be still compatible with the ProBond™ or Ni-NTA purification column.
- Increase stringency with NaCl and imidazole. Do an imidazole step gradient to elute the His-tagged protein.
- Decrease the pH by 0.5 –1.0 pH unit during denaturing purification. This can also be tried during native purification.
- Add beta-ME (beta-mercaptoethanol) to a maximum of 20 mM to reduce the disulfide bond.
- If too much resin is being used, the binding capacity exceeds the amount of His-tagged proteins. In this situation, endogenous proteins containing metal binding sites will bind to the resin with increased affinity.
- Co-elution of non-tagged proteins can be eliminated by doing a second round of purification from the eluate. Thus, dialyze against the binding buffer with 10–20 mM imidazole. This will significantly reduce the nonspecific binding capacity.
Try stringent purification methods including:
- Increase NaCl concentration to 2 M or greater. This should be tried first because the salt can be easily removed via dialysis.
- Increase the imidazole concentration
- Decrease the pH; for native purification, this can be reduced as long as protein function can be maintained. If not, decreasing pH should be tried anyway under such circumstances because protein can be refolded later if the native purification method does not achieve elution.
If the protein does not elute despite stringent elution procedure, the column must be stripped by one of the following methods:
- DTT: 1 mM or greater should be used.
- EDTA or EGTA: incubation of resin in 10–100 mM should be sufficient.
Note: Proteins should come off as long as they don’t precipitate on the column. Stripping the column will cause resin contamination of the protein. Either dialysis or centrifugation can remove the stripped resin. The stripped resin should not be reused again.
Consider an additional high stringency wash at a lower pH, i.e., between pH 6–4 prior to the elution. This applies for denaturing or hybrid purification. For native purification, the pH should only be decreased a little (exact pH depends on the pH optimum of the protein) in order to maintain protein structure.
The His-tagged protein may be an N-terminal tagged protein. Protein degradation during expression may not allow the expression of the full-length. If this is the case, an alternative is C-terminal His-tagged expression. If additionally, the expression is done in T7-promoter driven vector such as pET vector, induce with only 0.5 to 0.7 mM IPTG instead of 1 mM. Lastly, try to work at 4 degrees C at all times and use protease inhibitors during lysis.
The nickel in the column is reduced. Avoid using DTT, buffers with amines in solutions (Tris, HEPES, MOPS) and certain amino acids (arginine, glutamine, glycine). If the sample needs to be reduced, 20 mM beta-mercaptoethanol can be used.
In order to recharge the column, the Ni2+ must first be stripped away. We recommend incubating the column in 1M HCl for 1 hour at room temperature. At this point, the column should turn from brown to colorless. Wash with water to remove acid and add 50 mM NiSO4 to recharge the column. At this time, the column should return to its normal blue color.
A strong chelating agent like EDTA or EGTA has stripped the column and the Ni2+ has leached out. Use no more than 1 mM EDTA. We do not recommend reusing stripped resin.
pH drift is typical with these buffers. Adjust with concentrated HCl if the pH is too high or with 10 N NaOH if the pH is low.
ProBond™ and Ni-NTA beads can be used in FPLC columns. However, the beads can only withstand low pressure (~2.8 psi max).
Removal of Fusion Tags from Recombinant Proteins
Here are a few suggestions:
- Make sure that the cleavage sequence is present
- Try denaturation to expose the cleavage sites
- Increase the amount of enzyme for cleavage
- Modify the tag and cleavage site to contain linkers
The enzyme could be denatured. Try buffer exchange or dialysis before digestion with EKMax™ Enterokinase.
For Research Use Only. Not for use in diagnostic procedures.