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Anti-Protein C Affinity Matrix Troubleshooting

Product No. 11815024001

Troubleshooting

Little or no protein C tagged protein is eluted

  • Tagged protein is degraded
    → Include protease inhibitors and perform purification at +2 to +8 °C.
  • Tagged protein not fully eluted
    → If working at less than +37 °C, increase temperature, time, and/or number of elutions. → Try batch mixing of peptide solution with matrix.
  • Tagged protein expression is absent
    → Check for expression of protein in crude extract by western blot or biochemical assay.
  • Tagged protein expression is very low
    → Load larger volume of extract. Run column several times, pool, and concentrate final eluates.

Large quantities of tagged protein remain in the flow-through sample

  • Column is overloaded
    → Decrease amount of loaded protein extract.
  • Column not regenerated after use
    → Regenerate column.

Column flow stops

  • Column is overloaded
    → Decrease amount of loaded protein extract.
  • Starting extract contains insoluble materials
    → Preclear starting extract by high-speed centrifugation or filtration.
  • Air bubble in needle
    → Replace needle or place gentle pressure on column by briefly covering top of column with gloved hand.

Tagged protein appears degraded (a smear or multiple lower molecular weight bands on western blot)

  • Protease activity during procedure
    → Increase protease inhibitors in protein extract sample. Perform all steps at +2 to +8 °C.

Detection of Ig heavy or light chain on western blot following immunoprecipitation

  • Small quantities of heavy or light chain will accumulate over time in the matrix supernatant. This does not affect the performance (capacity) of the matrix before its expiration date.

→ Pre-rinse affinity matrix before using for immunoprecipitation.

Materials

     
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