Bio-Rad Chromatography Standards - Manual (Page 3)

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. The mixture
contains thyroglobulin,
γ
-globulin, ovalbumin, myoglobin, and vitamin
B
12
. Both vitamin B
12
and myoglobin are visible when applied to a
column and provide a means of ensuring that the column is properly
packed and that the sample is eluting evenly. The standard is supplied
as a set of 6 vials. Each vial of the lyophilized mixture is reconstituted
with 0.5 ml deionized water prior to use. The total protein content is
approximately 18 mg per vial.
1.1 Instructions
Rehydrate the vial by adding 0.5 ml of deionized H
2
O. Swirl gently to
mix and allow the vial to stand in ice for 2–3 min. Swirl the vial again
and apply the appropriate volume of standard to the column (see
following recommendation). The hydrated mixture should be kept at
2–8°C, for up to two weeks. For HPLC applications, the standard
should be centrifuged before application to remove any fine particulates.
Table 1. Gel Filtration Standard Components
Amount
Component Molecular Weight* per Vial (mg)
Thyroglobulin (bovine) 670,000 5.0
γ - globulin (bovine) 158,000 5.0
Ovalbumin (chicken) 44,000 5.0
Myoglobin (horse) 17,000 2.5
Vitamin B
12
1,350 0.5
18
*Estimates of molecular weights from:
1. CRC Handbook of Biochemistry, (Sober HA., ed.) Chemical Rubber
Company, Cleveland, Ohio (1968).
2. Merck Index, 9th edition, (Windholz M, ed.) Merck and Company, Inc.,
New Jersey (1976).
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