Trouble Shooting - His Tag Protein Purification

Possible Cause
No binding
The column is not equilibrated sufficiently in the buffer.
Repeat or try a longer equilibration step.
Protein precipitation in the column filter.
Clean the column, exchange or clean the filter.
Purification buffers contain chelating agents such as EDTA or EGTA.
Prepare new buffer without chelating agents.
Histidine tag is buried within the protein and unavailable to bind under native conditions.
Use another fusion tag, or try fusing the histidine tag to the other end of the protein.
Low yield
The concentration of imidazole in purification buffer and washing buffer is high. Binding affinity to the resin is weak.
Optimize imidazole concentration to be specific to your protein.
Degraded by proteases.
Add protease inhibitors.
Protein is still attached to ligand.
Try a higher concentration of imidazole.
High flow rates may not allow sufficient time for the proteins to bind and can be lost in the flow through.
Flow rate is critical to protein binding. Use lower flow rate, or try batch binding.
Overloading the column can reduce the protein yield.
The recommended rule is  to keep the protein load  within 50–75% of the column volume.
Host proteins contain multiple tandem histidine residues, they can bind weakly to the nickel resin and co-elute with the protein of interest.
Low concentration of imidazole can be added to the lysis and wash buffers to elute the nonspecific binding protein.
Host chaperone protein (HCP) is a common contaminant.
MgCl2 and ATP can be used to remove the chaperone proteins.

Trouble Shooting - GST Tag Protein Purification

Probable Cause
The yield of the purified GST fusion protein is low or undetectable.
The fusion protein forms inclusion bodies.
Grow bacteria at a lower temperature (20-30°C), or reduce final concentration of IPTG to 0.1 mM for protein induction, or reduce the induction time.
Properly dissolve and refold the inclusion body prior to the purification.
The fusion protein does not bind to Glutathione Resin efficiently.
Use batch method for purification.  Incubate clear solution (then sonicate, etc.) containing GST-fusion protein with Glutathione Resin for 2 hours or longer (i.e. overnight) and then load the mixture onto the column.
The fusion protein does not contain active GST.
Use mild sonication condition or other lysis method, such as lysozyme so that GST is not denatured.
The fusion protein is degraded by protease.
Add appropriate protease inhibitors such as PMSF in the lysis solution and wash solution.
The fusion protein is not efficiently eluted from Glutathione Resin.
Increase elution time or increase the concentration of glutathione to 15 mM or higher in the elution buffer.
Adjust the pH of the elution buffer to 8.0-9.0 without increasing the glutathione concentration.
Add Triton X-100 (0.1%, final concentration) or n-Octylglucoside (2%, final concentration) or NaCl (0.1-0.2 M, final concentration) to the elution buffer.
Multiple bands observed in the eluted protein
The fusion protein is degraded by protease.
Add appropriate protease inhibitors (or inhibitor cocktails) such as PMSF in the lysis solution and wash solution.
Some host proteins, such as chaperonins, may interact with the fusion protein.
Add DTT (5 mM, final concentration) in the wash buffer.  Incubate the recombinant protein solution in chaperonin buffer (2 mM ATP, 10 mM MgSO4, 50 mM Tris-HCl) at 37°C for 10 min prior to the purification.
Over-sonication will cause some protein to bind to the fusion protein.
Use milder sonication condition or another lysis method.
Some protein will bind to the fusion protein or beads non-specifically.
Optimize the wash conditions.  Detergents such as 1% Triton X-100, 1% Tween-20, 0.03% SDS, or 0.1% NP-40 may be used to reduce non-specific binding.  Salt concentration in the wash solution can also be optimized to reduce non-specific binding.

Trouble Shooting – Antibody Purification

Possible Cause
The flow rate of the column is very low (<0.5 ml/minute).
Tiny air bubbles from buffer or particles from sample block the gel pores.
De-gas buffers and samples.  Do not allow the column to dry.
A considerable amount of sample has been loaded, but no specific antibody of interest is detected.
The concentration of the antibody of interest is very low.
Purify the antibody using the specific antigen coupled to a resin (i.e., High-Affinity Iodoacetyl Resin, Cat. No. L00403).
The antibody is degraded.
The antibody is sensitive to low-pH
elution buffer.
Neutralize the eluted fractions with Neutralization Buffer immediately after elution.
No antibody is detected in any elution fraction.
The IgG subclass does not bind to this resin.
Try other affinity chromatography media to purify the antibody, such as Protein G Resin or Protein L Resin.

Trouble Shooting - Immunoprecipitation

Possible Cause
High background
Incomplete washing.
Increase the number of washing steps and prolong the washing steps. Background can be avoided using GenScript MagBeads.
Protein degradation during IP.
Add fresh-prepared protease inhibitors when sample is lysed.
Non-specific binding
The structure of agarose beads will absorb non-specific protein.
Pre-incubate agarose beads with the lysate before immunoprecipitation. Using  GenScript's MagBeads will eliminate this step.
No target protein detected
Expression level of target protein is low in the sample.
Increase the amount of lysate.  

FAQ - eStain® 2.0 Protein Staining System

FAQ - eBlot Protein Transfer System



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