Spin Columns

Figure 2.1B illustrates a miniature GPC chromatographic column with a frit on the end used as a spin column. These miniature columns are commercially available in different sizes (Harvard Apparatus, Holliston, Mass.) and can be formatted as a microtiter plate consisting of an array of 96 miniature columns. Likewise, similarly sized miniature fritless spin columns are available with holes at the bottom of the column that are smaller than the diameter of the GPC media particles, thereby reducing possible sample and protein losses due to adherence to the frit (Glygen Corp., Columbia, Md.). Larger spin columns are also available (Pharmacia, BioRad). Miniature 96 GPC spin column arrays can be easily prepared with a Millipore (Danvers, Mass.) multiscreen filtration system containing a hydrophilic Durapore filter (with a pore diameter of 0.65 mm) at the bottom of each well [15, 16]. The system has a 96-well collection plate for the spin column eluate and samples can be either directly applied at the top of each column or preferably loaded simultaneously into all the columns using a 96-well top plate with pinholes (MDS Protana, Denmark) for transfer to the 96 columns upon cen-trifugation [16]. Commercially prepared 96-well size exclusion microplates are also available [AutoSeq96, GE Healthcare (Amersham Biosciences); SigmaSpin, Sigma-Aldrich].

74 I 2 Drug Screening Using Gel Permeation Chromatography Spin Columns Coupled with ESI-MS Table 2.1 GPC gel types and fractionation ranges.

Polyacrylamide (BioRad)

Type Fractionation Range

P2 100-1,800 Da

P4 800-4,000 Da

P6 1,000-6,000 Da

P10 1,500-20,000 Da

P30 2,500-40,000 Da

P60 3,000-60,000 Da

P100 5,000-100,000 Da

Sephadex (Pharmacia)

Type Fractionation Range

G10 <700 Da

G25 1,000-5,000 Da

G50 1,500-30,000 Da

G75 3,000-80,000 Da

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