Streptavidin Mutein Matrix Troubleshooting

Product No. 03708152001

Troubleshooting

Biotinylated protein appears in flow-through

Column overloaded
Reduce sample load.

Binding conditions were not optimal
In some cases, monobiotinylated proteins may require a higher salt concentration to bind to the mutant streptavidin. Increase ammonium sulfate concentration to 0.6–1 M; ensure the protein is not precipitated! Also, wash matrix (following sample application) with Equilibration Buffer that contains 400 mM ammonium sulfate to minimize the loss of monobiotinylated protein during washing. Check sample pH before loading onto the column; pH after sample preparation should be approx 7.2. Increase the time allowed for the sample to bind to the matrix.

Matrix was not efficiently regenerated and/or equilibrated from the previous purification.
Repeat regeneration and equilibration step.

Column was not properly packed, leading to cracked gel bed (sample channeling)
Repeat column packing; avoid gel bed cracking.

Concentration of free biotin from biotinylation step was too high, and was thus efficiently competing with biotinylated protein
Check biotinylation protocol; biotin concentrations in the range of the AviTag biotinylation reaction (150 M) are not critical for binding. Higher concentrations of free biotin should be avoided; reduce free biotin concentrations via dialysis or ultrafiltrate the protein sample.

Incomplete biotinylation reaction, nonbiotinylated protein in flow-through
Perform a Western blot with SAPOD staining; ensure proper biotinylation conditions.

Biotin residue of biotinylated protein was not freely accessible for binding to mutant streptavidin
Vary binding conditions (reduce or increase total ionic strength). Change biotinylation protocol. Change AviTag position (C- versus N-terminus).

Biotinylated protein appears in wash fraction

Stable binding of biotinylated protein may need a continuously higher ammonium sulfate concentration even in the wash step.
Use Equilibration Buffer that contains 400 mM ammonium sulfate for the wash step; if needed, increase the ammonium sulfate concentration to 800 or 1000 mM.

Purified protein in eluate is not homogenous

Protein degradation during preparation and/or purification.
Add protease inhibitors during protein preparation; perform purification at +2 to +8 °C.

Internal initiation sites or premature stop may lead to shortened proteins.
Check and correct for internal initiation sites.

Nonspecific interaction of other proteins in the biotinylation mixture with the matrix.
Check with a Western blot and Streptavidin-Peroxidase staining; reduce the
concentration of ammonium sulfate in the sample and column preparation step.

Materials

     
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