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1. Filters with low protein-binding properties and pore size of 0.22 pm (see Note 4).

2. Protein standards: five proteins with MMpp spanning the range of the expected MMpp of the sample to be analyzed (see Note 5).

Table 1

Sample and Column Requirements for SEC-UV/LS/RI Analysis

Optimal amount of protein

Table 1

Sample and Column Requirements for SEC-UV/LS/RI Analysis

Optimal amount of protein

MM

MM

MM

MM

Column0

>200 kDa

40-200 kDa

10-40 kDa

<10 kDa

Superose 6 HR 10/30

50

50-100

Not

Not

suitable

suitable

Superdex 200 HR 10/30

50

50-100

100-200

Not

suitable

Superdex 75 HR 10/30

Not

50-100

100-200

Not

suitable

suitable

Superdex peptide HR 10/30

Not

Not

Not

400-800

suitable

suitable

suitable

a the Superose/Superdex columns from the HR 10/30 series were extensively tested by the author in a variety of buffer conditions (including buffers supplemented with various detergents or denaturants) as suitable for the SEC-UV/LS/RI approach; shown in bold type are the optimal column matches for a given MM range (as expected for a given oligomeric state). SEC, size-exclusion chromatography; UV, ultraviolet; LS, light scattering; RI, refractive index; MM, molar mass.

a the Superose/Superdex columns from the HR 10/30 series were extensively tested by the author in a variety of buffer conditions (including buffers supplemented with various detergents or denaturants) as suitable for the SEC-UV/LS/RI approach; shown in bold type are the optimal column matches for a given MM range (as expected for a given oligomeric state). SEC, size-exclusion chromatography; UV, ultraviolet; LS, light scattering; RI, refractive index; MM, molar mass.

3. Buffer: aqueous buffer compatible with the SEC column requirements; for the majority of SEC media, 150 mM salts must be present to prevent electrostatic interactions with the column's matrix (for the analysis of a-HL and LamB: 20 mM HEPES, 200 mM NaCl, 1 mM ethylenediamine tetraacetic acid [EDTA]; pH 7.4, 5 mM L-glutamic acid, 2 mM (0.05%) dodecyl maltoside, C12M). The buffer should be filtered through a 0.1-|im filter.

4. Sample: 50-500 |g of protein sample (see Table 1 for guidelines regarding optimal sample amounts) in a volume that corresponds to less than 3% of the total volume of the SEC column (with 1% being the preferred loading volume).

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