Bio-Rad Unstained SDS-PAGE Standards User Manual

Ordering Information
Catalog Number Product Description
Molecular Weight Standards
161-0303 SDS-PAGE Standards, High, 200 µl 161-0304 SDS-PAGE Standards, Low, 200 µl 161-0317 SDS-PAGE Standards, Broad, 200 µl 161-0314 Silver Stain SDS-PAGE Standards, Low, 200 µl 161-0315 Silver Stain SDS-PAGE Standards, High, 200 µl 161-0306 Biotinylated SDS-PAGE Standards, Low, 250 µl 161-0311 Biotinylated SDS-PAGE Standards, High, 250 µl 161-0319 Biotinylated SDS-PAGE Standards, Broad, 250 µl 161-0320 2-D SDS-PAGE Standards 161-0326 Polypeptide SDS-PAGE Standards, 200 µl
Prestained Standards
161-0305 Prestained SDS-PAGE Standards, Low, 500 µl 161-0309 Prestained SDS-PAGE Standards, High, 500 µl 161-0318 Prestained SDS-PAGE Standards, Broad, 500 µl 161-0324 Kaleidoscope Prestained Standards, 500 µl 161-0325 Kaleidoscope Polypeptide Standards, 500 µl
IEF Standards 161-0310 IEF Standards, pI range 4.45-9.6, 250 µl
Silver Stain
SDS-PAGE Standards,
Low Range
Catalog Number
161-0314
Product shipped on dry ice.
Store at -20 °C upon arrival.
Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules CA 94547
4006047 Rev C
SDS-PAGE Molecular Weight Standards,Low Range Specifications
Low Range
Range Contents Rabbit muscle phosphorylase b
Volume 200 µl concentrated solution Storage -20 °C Shelf Life 1 year at -20 °C Applications 400 full size gels
per vial 800 mini gels Recommended 12.5%*
gel percentages
14,400 to 97,400 daltons
Bovine serum albumin Hen egg white ovalbumin Bovine carbonic anhydrase Soybean trypsin inhibitor Hen egg white lysozyme
*Note:
The lowest recommended gel percentage for the low range standards is 10%. At gel percentages of 10% or less, one or more of the standards may migrate at the dye front, depending on running conditions and other factors. As a result, only four or five bands may be visible after staining. If this should occur, an increase in gel percentage is recommended.
Silver Stain SDS-PAGE Standards contain approximately 700 µg total protein in 50% glycerol (w/v), 300 mM NaN3, 20 mM Tris, and 4 mM EDTA. The proteins have been blended to give bands of equal intensity on SDS polyacry­lamide gel systems run according to Laemmli1and stained with Bio-Rad Silver Stain or Silver Stain Plus. Different results may be obtained when alternative silver staining chemistries are used.
Reference
1. Laemmli, U. K., Nature, 227, 680 (1970).
2. Hames, B. D. and Rickwood, D., Gel Electrophoresis of Pro- teins: A Practical Approach, Second Edition, p. 17, Oxford University Press, New York (1990).
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Protocol
100
90 80 70 60 50 40 30 20 10
Daltons
12108642 cm
Dilute standards 1:20 in SDS Reducing Sample Buffer.* Heat for 5 minutes at 95 °C. Cool and load 10 µl/well for full length gels (16-20 cm) or 5 µl/well for mini gels. These load volumes and dilutions have been optimized for development with Bio-Rad Silver Stain or Silver Stain Plus for approxi­mately 10 minutes. If silver stain development times vary, the loading volume or dilution of the standards may need to be adjusted to optimize the band intensity.
* SDS Reducing Sample Buffer (Prepare imme-
diately before use)
ß-mercaptoethanol 25 µl Stock sample buffer 475 µl
500 µl
Stock Sample Buffer (Store at room temperature)
Distilled water 4.8 ml
0.5M Tris-HCl, pH 6.8 1.2 ml Glycerol 1.0 ml 10% (w/v) SDS 2.0 ml
0.1% (w/v) bromophenol blue 0.5 ml
9.5 ml
Use of stock sample buffer with insufficient or old ß-mer­captoethanol may result in doublets at the soybean trypsin inhibitor and ovalbumin bands.
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Protein Molecular Weights
Molecular
Protein Weight References
Rabbit muscle 97,400 Titani, K., et. al., Proc. Natl. Acad. Sci. phosphorylase b USA, 74, 11, 4762 (1977).
Bovine serum 66,200 Brown, J. R., Fed. Proc., 34, 591 (1975). albumin (BSA)
Hen egg white 45,000 Warner, R. C., "Egg Proteins," in: The ovalbumin Proteins, Vol. IIA, p. 435 (Neurath, H.
and Bailey, K., eds.), Academic Press, New York (1954).
Bovine carbonic 31,000 Davis, R. P., "Carbonic Anhydrase," in: anhydrase The Enzymes, Vol. V, p. 545, (Boyer,
P. D. eds.) Academic Press, New York (1971).
Soybean trypsin 21,500 Wu, Y. V. and Scheraga, H. A., Bio-chem- inhibitor istry, 1, 698 (1962).
Hen egg white 14,400 Jolles, P., Angew. Chem., Intl. Edit., 8 227 lysozyme (1969).
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Phosphorylase b Bovine serum
album Ovalbumin
Carbonic anhydrase
Soybean trypsin inhibitor
Lysozyme
Fig. 1. Silver Stain SDS-PAGE Standards give bands of even intensities when stained with Bio-Rad's Silver Stain or Silver Stain Plus. Note that no extraneous
bands are present. Low range standards run on a 12.5% SDS polyacrylamide gel and stained with Bio-Rad's Silver Stain Kit.
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Fig. 2. Curve generated by plotting the molecular weight of the low range molecular weight standards run on a 12% SDS polyacrylamide gel vs. the dis­tance migrated from the interface of the stacking and separating gels in centimeters. An alternative method
is to plot the log10relative mobility (Rf) vs. the gel con­centration, %T, (percentage total monomer, i.e. grams acrylamide plus bis acrylamide/100ml).
distance migrated by protein
Rf=
distance migrated by dye
The curve can be used to determine molecular weights of
unknown proteins.
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