Amicon centricon concentrators

A solution of B800-depleted
LH2 in a mixed buffer of 20 mM Tris and 10 mM succinate containing
0.1% DDM (pH 8.0) was mixed with 1/100 volume of a methanol solution
of AcChl a or BChl a, followed by
incubation at 35 °C for 2 h in the dark. The sample was concentrated
by ultrafiltration using Amicon centricon concentrators (30 kDa cutoff,
Merk Millipore Ltd.) and was loaded onto a Sephacryl-S200 column (GE
Healthcare) in 20 mM Tris buffer containing 0.1% DDM and 150 mM NaCl
(pH 8.0). LH2 proteins collected were desalted by ultrafiltration
using Amicon centricon concentrators (30 kDa cutoff, Merk Millipore
Ltd.). LH2 proteins, which are reconstituted with AcChl a and BChl a, are hereafter denoted as AcChl-reconstituted
LH2 and BChl-reconstituted LH2, respectively.
The occupancy
of AcChl a in the B800 sites in AcChl-reconstituted
LH2 was estimated from electronic absorption spectra of extracted
chlorophyllous pigments in the Q_y region
in methanol, as reported elsewhere.^{19 (link)} The
occupancy of BChl a in the B800 sites in BChl-reconstituted
LH2 was estimated by comparing Q_y absorbance
of B800 BChl a in BChl-reconstituted LH2 with that
in native LH2.^{19 (link)}