The protein solution may not be flowing through the column due to one or more of the following:

• Centrifugation speed was too low.

  Check the centrifuge to ensure that it is capable of generating 6,700 x g. Sufficient centrifugal force is required to move the liquid phase through the resin.

• Protein solution is too viscous.

  Dilute the protein solution as described in the protocol.

• Cellular debris is present in the protein solution.

  Filter the sample using a 0.45 µM filter or spin down insoluble materials and transfer the liquid portion to the column. Solid, insoluble materials can cause severe clogging problems.

• Inadequate spin time.

  Spin for an additional minute to ensure that the liquid is able to flow completely through the column.

Insufficient depletion of major proteins may be caused due to:

• Overloading the column with proteins.

  Decrease the amount of serum or plasma that is loaded onto the column.

• Improper sample preparation.

  Ensure that the serum or plasma sample is properly prepared by diluting it in the provided Wash Solution G.

The eluted protein may be degraded due to one or more of the following:

• Eluted protein solution was not neutralized.

  Add 18.6 µL of Protein Neutralizer to each 200 µL of eluted protein in order to adjust the pH to neutral. Some proteins are sensitive to high pH, such as the Elution Buffer C at pH 12.5.

• Eluted protein was not neutralized quickly enough.

  If eluted proteins are not used immediately, degradation will occur. We strongly suggest adding Protein Neutralizer in order to lower the pH.

• Proteases may be present.

  Use protease inhibitors during all steps of sample preparation, and during storage of the serum, if desired.

• Bacterial contamination of the protein solution.

  Prepare the serum samples with 0.015% sodium azide.

Low protein concentration in the elution can be caused by a low level of serum proteins present in the initial sample. Increase the amount of serum or plasma that is loaded onto the column. The input amount can be verified using a reliable colorimetric assay. We recommend starting with 600 μg to 800 μg of protein in input sample.