SEC Column Selection for Separating 20 kDa and 35 kDa Proteins

Record: 652

Category: Column Selection
Product: KW-802.5
Sample: Protein

Question

I am looking for a size-exclusion chromatography (SEC) column to separate a 35 kDa protein from a 20 kDa protein. I have previously used Shodex columns for monoclonal antibody analysis and was very satisfied with the performance.

Answer

Yes. For separating proteins in the 20–35 kDa molecular-weight range, Shodex offers silica-based aqueous size-exclusion chromatography (SEC) columns with pore sizes well suited to this separation. These columns are compatible with standard aqueous buffer systems and provide reliable resolution for closely spaced protein species in this molecular-weight region.

Shodex PROTEIN KW-802.5 is the primary recommendation, as it is designed for protein and enzyme analysis in the low-to-mid molecular-weight range and provides effective separation for proteins from approximately 5,000 to 100,000 Da. This makes KW-802.5 well suited for resolving 20 kDa and 35 kDa proteins with good SEC resolution.

When broader molecular-weight coverage is required, Shodex PROTEIN KW-803 may be selected, as it extends the usable range to higher molecular weights while maintaining separation performance in the 20–35 kDa region. Shodex PROTEIN LW-803 may also be considered when alignment with antibody or biologics SEC workflows is desired, as its usable molecular-weight range also encompasses proteins in this size range.

Recommended Applications:

• Standard Proteins (KW-802.5)
  Demonstrates SEC separation of protein standards in the molecular-weight range relevant to 20–35 kDa analysis.
• Standard Proteins (KW-802.5)
  Illustrates resolution and calibration behavior of KW-802.5 using common protein standards.
• SEC Analysis of Rituximab (LW-803)
  Example of protein and antibody SEC performance using LW-803.
• SEC Analysis of Antibody-Drug Conjugate (LW-803)
  Demonstrates high-molecular-weight protein analysis and SEC method setup.

Additional Comments

Final column selection depends on sample composition, buffer system, detector type, and required resolution. Flow rate and ionic strength may be optimized to improve separation between closely spaced molecular-weight species.

For more tailored recommendations, please contact Shodex Technical Support with additional method detail at support@shodexhplc.com.

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