Assay Buffers

• DLS is compatible with a range of buffers
• Use filtered buffers
• Measure scattering of buffer control
  • Detergents frequently have micelle size similar to that of proteins and scatter significantly

Samples

• DLS is very sensitive to small amounts of aggregates.
  • Significant aggregates will make accurate size measurements difficult.
  • Samples should be filtered or centrifuged (6000g, 10-30 min) prior to transfer to the clear bottom plate to remove large aggregates and precipitates.
• The minimum amount of sample required depends on the size of the molecule, as large molecules scatter more light than small molecules.
  • For most protein samples, concentrations of 0.2 mg/ml or higher are recommended.
  • As little as 0.125 mg/ml of lysozyme or 12.5 μg/ml of IgG can be detected.
• Small molecule aggregation analysis should be done at working concentrations in and working solvents (e.g. 5% DMSO).
  • Solvent controls are essential. DMSO itself is prone to aggregation.
• When working with membrane proteins, note that empty detergent micelles will not resolve from protein-filled micelles (except when empty micelle varies in size by 5-10X)
  • Avoid excess detergent by purifying membrane proteins by affinity and/or size-exclusion chromatography and avoid concentrating after purification

Generally, SEC-MALS data collection is done in ASTRA 7 with HPLC control.  

CMI SEC-MALS Getting Started Guide

CMI SEC-MALS Guide to Protein Conjugate Analysis, guide to measuring dn/dc values and performing protein conjugate data analysis.

CMI SEC-MALS Guide to Working with Custom Solvents, for solvents that cannot use PBS or water as the solvent model.

SEC-MALS Technology page from Wyatt Technologies.

• purified protein samples (must be filtered before loading)
• running buffer (1L)

• analytical SEC column (the CMI usually has 1-2 working SEC-MALS columns that users may borrow, if their samples are purified by SEC and in an appropriate buffer)

Assay Buffers

• Running buffer should always be chosen to be compatible with both the SEC column and the protein sample.
• Recommended Buffer: 25 mM HEPES pH 7-7.5, 150 mM NaCl (filtered).
• Make sure you know the buffer compatibility of the SEC column you are using.
  • Most silica columns will not tolerate pH above 7.5.
• Some buffer components (e.g. glycerol) will require customization of the solvent profile. Take note of the absolute refractive index of the solvent after equilibration and before data collection.

Samples

• The sample should be prepared in running buffer to minimize RI peak due to sample solvent.
  • This is particularly important for samples that run near the solvent peak.
  • It is good practice to run a size-exclusion chromatography purification prior to analytical SEC-MALS to buffer exchange and clear aggregates. This is a requirement if you are using a shared CMI column.
• Samples must be filtered or centrifuged prior to injection.
• Concentration should be accurately measured to assess column recovery.
  • Know the concentration (in mg/ml) of your protein.
  • Know the UV extinction coefficient (in ml/mg•cm) of your protein
• Protein aggregates can damage the column.
  • Filter or centrifuge samples before use.
  • Assess protein heterogeneity via dynamic light scattering (in the DynaPro plate reader).
  • Purify protein samples with soluble aggregates by size-exclusion chromatography.
• Recommended protein concentration varies depending on protein mass:
  • Scattering is proportional to mass
    • Larger proteins require less sample than smaller proteins.
  • Typical range 5 – 500 µg/injection
    • BSA (67 KDa) 100 µl at 2 mg/ml always gives a good light scattering signal
• Sample volume:
  • Maximum injection volume: 100 µl
  • Minimum injection volume: 5 µl
  • Glass autosampler vial with low volume insert has 10 µl dead volume.
    • Fill vial with at least 110 µl for a 100 µl injection.