CHARACTERIZATION OF sLR11 IN SERUM AND CSF BY
GEL FILTRATIONIt was dialyzed, lyophilized, and subjected to
gel filtration on a Superdex 75 HR 10/30 column (Amersham Biosciences) using an AKTA Purifier (Amersham Biosciences) in 10 mM [NH.sub.4][HCO.sub.3] buffer (pH 9.4).
Determination of native molecular weight by
gel filtrationBy combining
gel filtration chromatography of patient sera and PSA immunoreactivity experiments these authors identified a larger peak corresponding to a mass of 80-90 kDa and a smaller peak corresponding to a mass of 25-40 kDa.
The native molecular mass was estimated by
gel filtration chromatography on the Sephacryl S-200 column, according to the method of Andrews (1964).
To obtain the VLDLR fraction from the serum of a patient with type III hyperlipoproteinemia, we conducted ultracentrifugation and
gel filtration. Fig.
Gel filtration for measuring the molecular weights of native Hemolysin was carried out on a Sephacryl S-200 column (1.5 x 100 cm Pharmacia), which had been calibrated with the following standard proteins: bovine serum albumin (67 kDa), Galanthus nivalis agglutinin (48 kDa), trypsin (23.3 kDa), equine myoglobin (17 kDa), and cytochrome c (12.4 kDa).
The antibody was treated as mentioned above and transferred to a buffer containing 0.25 mol/L Bis-Tris buffer, pH 6.2, in a prepacked
gel filtration column containing Sephadex G-25, (PD-10 column, GE Healthcare).
To obtain high purity protein, it was further subjected to
gel filtration chromatography with a superose 12 HR 10/30 column.
Proteasomes were eluted from the resin by a linear increasing gradient of 0-500 mmol/L NaCl dissolved in TEAD buffer, precipitated by saturation of the solution with 80% saturated [(N[H.sub.4]).sub.2]S[O.sub.4], and then subjected to
gel filtration on Superose 6 as described above.
The crude fractions III-a and III-b were dissolved separately in 3.0 ml of 0.2 M NaCl and purified further by
gel filtration on 1.6 X 85 cm Sephacryl S-400 column (Amersham Biosciences) and eluted with 0.2 M NaCl.
Gel filtration chromatography analysis was also performed with the patient's serum, as shown in Fig.