Ordering Information
Catalog
Number Product Description
161-0319 Biotinylated SDS-PAGE Standards, Broad Range,250 µl
161-0306 Biotinylated SDS-PAGE Standards, Low Range, 250 µl
161-0311 Biotinylated SDS-PAGE Standards, High Range, 250 µl
161-0322 Biotinylated SDS-PAGE Standards Kit, Broad Range, AP*
161-0321 Biotinylated SDS-PAGE Standards Kit, Broad Range, HRP*
161-0307 Biotinylated SDS-PAGE Standards Kit, Low Range, HRP*
161-0308 Biotinylated SDS-PAGE Standards Kit, Low Range, AP*
161-0312 Biotinylated SDS-PAGE Standards Kit, High Range, HRP*
161-0313 Biotinylated SDS-PAGE Standards Kit, High Range, AP*
170-6528 Avidin-HRP, 2 ml
170-6533 Avidin-AP, 1 ml
170-6431 Horseradish Peroxidase Conjugate Substrate Kit
170-6432 Alkaline Phosphatase Conjugate Substrate Kit
* Each kit contains 250 µl Biotinylated Standards, 1 ml
Avidin-AP or 2 ml Avidin-HRP, and complete instructions.
Biotinylated SDS-PAGE
Standards, Low, High,
and Broad Range
Catalog Numbers
161-0306
161-031 1
161-0319
Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA 94547
LIT395 Rev D
Biotinylated SDS-PAGE
Standards, Low , High,
and Broad Range
Bio-Rad’s Biotinylated SDS-PAGE Standards are
a mixture of biotinylated proteins that can be used for
accurate molecular weight determinations of immune
detected proteins. The standards have been blended to
give equal intensities when detected with avidin-HRP
and HRP Color Development Reagent.
The Biotinylated SDS-PAGE Standards and the
sample proteins are run on an SDS polyacrylamide gel
and electrophoretically transferred to nitrocellulose,
PVDF, or Zeta-Probe®membranes. The protein samples and standards are exposed to the same reagents
during detection. This prevents distortions that may
occur if the standards lane is treated separately.
The molecular weights of the standards are not significantly altered by biotinylation. The consistent
molecular weights of the standards give accurate
molecular weight determinations every time without
increasing the number of steps or length of time of
immune detection.
1
Specifications
MW Ranges Low 14,400–97,000 daltons
Protein MW (daltons) Range Range Range
Myosin 200,000 X X
β-galactosidase 116,250 X X
Phosphorylase b 97,400 X X X
Bovine serum albumin 66,200 X X X
Ovalbumin 45,000 X X X
Carbonic anhydrase 31,000 X X
Soybean trypsin inhibitor 21,500 X X
Lysozyme 14,400 X X
Aprotinin 6,500 X
Contents Approximately 130 µg total protein in a buffer
Volume 250 µl
Storage -20 °C
Shelf life 1 year at -20 °C
Applications Dilute 1:4 for HRP color development; 60–100
High 45,000–200,000 daltons
Broad 6,500–200,000 daltons
Low High Broad
of 50% glycerol, 150 mM NaCl, 3 mM NaN
applications per vial
Dilute 1:20 for AP color development; 300–500
applications per vial
2
3
Protocol
1. When using HRP conjugates, dilute standards
1:4 in sample buffer.*When using AP conjugates,
dilute the standards 1:20 in sample buffer. Heat for
5 minutes at 95 °C. Cool and load 10 µl/well for
mini-gels. Load 10–15 µl/well for full length gels
(16–20 cm).
2. After electrophoretic blotting of the proteins,
detection of the biotinylated standards is performed after the blocking and primary antibody
incubation steps. The avidin conjugates are used in
a 1:3,000 dilution in antibody buffer (1% gelatin in
TTBS†). This solution should contain the appropriate dilution of blotting grade second antibody conjugate, protein A, or protein G conjugate. Incubate
the membrane 1 hour with gentle agitation at room
temperature.
3. Remove the conjugate solution, and wash the
membrane twice for 5 minutes in Tris buffered
saline with 0.05% Tween-20 (TTBS)†with gentle
agitation. Wash twice for 5 minutes in TBS.
3
4. Prepare the color development solution immediately
before use. Immerse the membrane in the solution.
Stop the development by washing the membrane in
distilled water for 10 minutes. Change the water at
least once during this time.
*
Sample buffer (SDS-PAGE reducing buffer)
Distilled water 4.0 ml
0.5 M Tris-HCl, pH 6.8 1.0 ml
Glycerol 0.8 ml
10% (w/v) SDS 1.6 ml
β-mercaptoethanol 0.4 ml
0.1% (w/v) Bromophenol blue 0.2 ml
8.0 ml
Note: Addition of a reducing agent such as BME is
important because there is no reducing agent in the
buffer as supplied.
†
Tris buffered saline (TBS)
(20 mM Tris, 500 mM NaCl, pH 7.5)
Tris base 4.84 g
NaCl 58.44 g
Dissolve Tris and NaCl in 1.8 L distilled water. Adjust
the pH to 7.5 with HCl, and adjust the volume to 2 L
with distilled water. For TTBS add 0.5 ml Tween-20 to
1 L of TBS (0.05% Tween-20).
4
Phosphorylase b
Bovine serum
albumin
Ovalbumin
Carbonic anhydrase
Soybean trypsin
inhibitor
Lysozyme
Myosin
β-galactosidase
Phosphorylase b
Bovine serum
albumin
Ovalbumin
Carbonic
anhydrase
Soybean trypsin
inhibitor
Lysozyme
Aprotinin
AB
Fig. 1. Biotinylated SDS-PAGE Standards, low and
broad range. A. Low range biotinylated SDS-PAGE stan-
dard run on a 12% gel, blotted to nitrocellulose, and
detected with Avidin-HRP. B. Broad range biotinylated
SDS-PAGE standards run on a 4–20% gradient gel, blotted to nitrocellulose, and detected with Avidin-AP.
5
Protein References
Protein Reference
Rabbit muscle myosin Woods, E. F., Himmelfarb, S.
E. coli
β-galactosidase Fowler, A. V. and Zabin, I.,
Rabbit muscle phosphorylase b Titani, K, et al.,
Bovine serum albumin (BSA) Brown, J. R.,
Hen egg white ovalbumin Warner, R. C., Egg Proteins, in
Bovine carbonic anhydrase Davis, R. P., Carbonic
Soybean trypsin inhibitor Wu, Y. V. and Scheraga, H. A.,
Hen egg white lysozyme Jolles, P.,
Bovine pancreatic trypsin Kassell, B. and Laskowski, M.,
inhibitor (aprotinin)
and Harrington, W. F.,
Chem.
, 238, 2374 (1963).
Nat. Acad. Sci. USA
(1977).
Sci. USA
, 74, 4762 (1977).
(1975).
The Proteins, Vol. llA, p. 435,
(Neurath, H. and Bailey, K. eds.)
Academic Press, New York
(1954).
Anhydrase, in: The Enzymes,
Vol. V, p. 545 (Boyer, P. D. ed.)
Academic Press, New York
(1971).
Biochemistry
Edit.
, 8, 227 (1969).
Biochem. Biophys. Res. Comm.
20, 463 (1965).
, 74, 1507
Proc. Nat. Acad
Fed. Proc.,
, 1, 698 (1962).
Angew. Chem., Intl.
6
J. Biol.
Proc.
34, 591
,
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