Preparation
This antibody was produced from a hybridoma resulting from the fusion of a mouse
myeloma with B cells obtained from a mouse immunized with purified, E. coli-derived
recombinant human beta-Catenin (GenBank accession # NM_001904). The IgG fraction of
the tissue culture supernatant was purified by Protein G chromatography. Beta-Catenin is a
cadherin-associated protein that is involved in the regulation of cell adhesion. It is also
involved in the canonical Wnt signal transduction pathway.
Formulation
Lyophilized from a 0.2 μm filtered solution in phosphate-buffered saline (PBS) with
5% trehalose.
Reconstitution
Reconstitute with sterile PBS to achieve a final concentration of 500 μg/mL.
Storage
Lyophilized samples are stable for twelve months from date of receipt when stored at -20° C
to -70° C. Upon reconstitution, the antibody can be stored at 2° - 8° C for 1 month without
detectable loss of activity. Reconstituted antibody can also be aliquotted and stored frozen
at -20° C to -70° C in a manual defrost freezer for six months without detectable loss of
activity. Avoid repeated freeze-thaw cycles.
Specificity
The antibody was selected for its ability to detect endogenous human, mouse and rat
beta-Catenin in Western blots.
Application
Western blot - An antibody concentration of 2.0 μg/mL is recommended.
Optimal dilutions should be determined by the individual laboratory.
Protocols for Immunoblotting:
Western blotting
Blotting buffer Blocking Solution Antibody Solution
25 mM Tris, pH 7.4
0.15 M NaCl
0.1% Tween 20
2% nonfat dry milk in
blotting buffer
Adjust pH to 7.4
2% nonfat dry milk in
blotting buffer
Adjust pH to 7.4
1. Transfer the electrophoresed proteins to Immobilon-P membrane (Millipore)
and incubate the membrane for 1 hour at room temperature in Blocking
Solution.
2. Incubate the membrane overnight at 4° C in antibody solution containing 2.0 μg/mL
mouse anti-human/mouse/rat beta Catenin.
3. Wash the membrane at room temperature for 30 minutes with 3 or more
changes of Blotting Buffer. Changing the membrane containers often reduces
background.
4. Incubate the membrane at room temperature for 1 hour in Antibody Solution
containing a 1:2,000 dilution of HRP-conjugated sheep anti-mouse IgG
(Amersham).
5. Wash the membrane for 1 hour with 5 or more changes of Blotting Buffer.
6. Detect with WesternGlo Chemuluminescent Detection Substrate (R&D
Catalog # AR004).
Cell lysates for Western blottings: To prepare total cell lysates, cells are
solubilized in hot 2x SDS gel sample buffer (20 mM dithiothreitol, 6% SDS, 0.25 M
Tris, pH 6.8, 10% glycerol, 10 mM NaF and bromophenyl blue) at
2 x 106 - 1 x 107 cells per mL. The extracts are heated in a boiling water bath for
5 minutes and then sonicated with a probe sonicator with 3 - 4 bursts of
5 - 10 seconds each. Samples are diluted with 1x SDS sample buffer to the
desired concentration.