Lit102E.
Lit102E.qxd 09/13/2002 2:35 PM Page i Table of Contents Section 1 1.1 1.2 1.3 1.4 Section 2 Introduction....................................................... 1 Instructions................................................................. Recommended Volume of Standard........................... Shelf Life..................................................................... Storage....................................................................... 1 2 5 5 Ordering Information...................
Lit102E.qxd 09/13/2002 2:35 PM Page 1 Section 1 Introduction Bio-Rad’s gel filtration standard is a lyophilized mixture of molecular weight markers ranging from 1,350 to 670,000 daltons. It is a calibration standard for gel filtration/size exclusion chromatography (SEC) columns used in protein purification and analysis under nondenaturing conditions, and is appropriate for gels with exclusion limits approximately 60,000 to 5,000,000 daltons.
Lit102E.qxd 09/13/2002 2:35 PM Page 2 1.2 Recommended Volume of Standard The general recommendation is to load no more than 1% of the column volume to avoid peak broadening effects. To achieve an absorbance of about 0.1 to 0.2 OD at 280 nm with various column sizes, use a volume of sample proportional to the total column volume. For example, a column of 170 ml total volume would require about 0.5 ml of standard, while a column of 40 ml would require about 0.125 ml.
Lit102E.qxd 09/13/2002 2:35 PM Page 3 F A&B E C A280 D Eluent volume F a. Bio-Sil SEC 125 column E C D A280 A&B Eluent volume F b. Bio-Sil SEC 250 column E CD A280 B A Eluent volume c. Bio-Sil SEC 400 column Fig. 1. Separation of standard on HPLC size exclusion columns.
Lit102E.qxd 09/13/2002 2:35 PM Page 4 Fig. 1. a. Bio-Sil SEC 125 column (exclusion limit* ≈ 80,000). b. Bio-Sil SEC 250 column (exclusion limit ≈ 300,000). c. Bio-Sil SEC 400 column (exclusion limit ≈ 800,000). Peaks: A. Protein aggregates (void peak) B. Thyroglobulin C. γ-globulin D. Ovalbumin E. Myoglobin F. Vitamin B12 Conditions Buffer Column Flow rate Sample Chart speed Detection 0.05 M Na2HPO4, 0.05 M NaH2PO4, 0.15 M NaCl, pH 6.8 300 x 7.8 mm 1.0 ml/min 20 µl 1 cm/min UV @ 280 nm; 1.
Lit102E.qxd 09/13/2002 2:35 PM Page 5 1.3 Shelf Life The lyophilized standard is stable for at least 1 year when stored at 2 to 8°C. The shelf life may be extended if stored at a lower temperature. 1.4 Storage The lyophilized standard may be stored at 2 to 8°C or lower. The reconstituted standard should be aliquoted and frozen at 0°C or lower and is stable for 5 years.
Lit102E.qxd 09/13/2002 2:35 PM Page 6 Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
