Clarification of Samples (< 1 mL) in Spin Filters (Nanosep® Centrifugal Devices) 2.5.3

Application Guidelines for Clarification of Samples (< 1 mL) in Spin Filters (Nanosep Centrifugal Devices)

Small-volume samples (< 1.0 mL) can be clarified by microporous surface filtration in the Nanosep MF centrifugal devices. MF filters are available in two pore sizes, 0.45 and 0.2 μm; and in two different membrane chemistries, Bio-Inert®, a modified low protein binding nylon surface; and GHP, a hydrophilic modified polypropylene surface. The centrifugal MF device family properties are summarized in Table 2.58. Typical applications of micro-volume MF clarification in proteomics include:

• Clarification of thawed plasma or serum that may contain cryoprecipitate material.
• Prefiltration of samples prior to injection into an HPLC system to remove fine colloidal material that could block a small particle diameter column.
• Prefiltration of samples prior to 1D and 2D electrophoresis to remove aggregated or insoluble material prior to analysis.
• Filter sterilization of components or additives for cell culture that are heat labile.

Table 2.58

Properties of the Nanosep® MF Centrifugal Devices

Property	Parameter
MF Filtration Membrane	Bio-Inert® is a modified nylon  GHP is a hydrophilic polypropylene
Filtrate Receiver	Polypropylene
Effective Membrane Area	0.28 cm2
Dimensions  Overall Length (with Cap)	4.5 cm (1.8 in.)
Capacities      Maximum Sample Volume      Final Retentate Volume      Final Receiver Volume      Hold-up Volume (Membrane/Support)	0.5 mL 0.5 mL 0.5 mL < 0.005 mL
Operating Temperature Range	0–40 °C (32–104 °F)
pH Range	3–14
Maximum Centrifugal Force	14,000 x g
Centrifuge	1.5 mL microcentrifuge rotors
Sanitization	70% ethanol

Protocol for Clarification of Samples (< 1 mL) in Spin Filters (Nanosep® Centrifugal Devices)

A. Materials Required

1. Nanosep MF centrifugal devices with Bio-Inert® or GHP membranes with a collection tube. For specifications, see Table 2.58.
2. Extra collection tubes for the Nanosep MF device
3. Degassed high purity water or buffer, such as phosphate biffered saline (PBS)

B. Nanosep MF Protocol for Working with Samples < 1 mL

For MF filtration with 0.45 or 0.2 μm pore sizes, samples should not be too heavily loaded with suspended solids or be too highly viscous, as this will severely impact the MF filtration flux in these limited area filter devices. In many cases, the filter will plug and will only pass a very small volume of filtrate. If necessary, prefiltration with a depth filter or sample dilution may be required. For example, for clarification of thawed frozen plasma or serum prior to abundant protein depletion, samples should be diluted 1:4 in PBS. Depending on the amount of turbidity of the thawed sample, it may be necessary to prefilter with a GF or Serum Acrodisc® syringe filter before using the 0.45 or 0.2 μm centrifugal MF filters.

1. Select the Nanosep MF device with a 0.45 or 0.2 μm pore size.
   Tip: For most applications, 0.45 μm pore size filters are sufficient to remove particulate material prior to chromatography applications. If removal of microorganisms is required, then the 0.2 μm pore size filter is recommended.
2. Add 0.5 mL of the sample and centrifuge at 14,000 x g for 5–10 minutes depending on the pore size membrane used.
3. If there is sample left in the retentate cup, mix and repeat Step 2.
4. If sample appears to be flowing through the filter very slowly, consider a prefiltration step.
5. Transfer the filtrate into a clean tube. Confirm that the protein sample of interest has been recovered in the filtrate with acceptable recovery. If not, the sample may have become aggregated and retained on the MF filter.
6. Recover the sample from the Nanosep MF retentate cup by adding PBS and mixing as a first step. If the retained material is still not recovered, it may be necessary to add solubilization agents, such as detergents (CHAPS, Triton^♦ X-100), chaotropic agents (urea or thiourea) or denaturants (guanidine HCl).

Ordering Information for Clarification of Samples (< 1 mL) in Spin Filters (Nanosep® Centrifugal Devices)

Nanosep MF Centrifugal Devices with Bio-Inert® Membrane

Part Number	Description	Pkg
ODM02C33	0.2 μm, aqua	24/pkg
ODM02C34	0.2 μm, aqua	100/pkg
ODM02C35	0.2 μm, aqua	500/pkg
ODM45C33	0.45 μm, wildberry	24/pkg
ODM45C34	0.45 μm, wildberry	100/pkg
ODM45C35	0.45 μm, wildberry	500/pkg

Nanosep MF Centrifugal Devices with GHP Membrane

Part Number	Description	Pkg
ODGHPC34	0.45 μm, clear	100/pkg
ODGHPC35	0.45 μm, clear	500/pkg

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