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1. Load cell extracts and separate proteins on an appropriate percentage
SDS polyacrylamide gel (SDS-PAGE) (smaller proteins should be run on a higher percentage gel and
higher molecular weight proteins should be run on a lower percentage gel). | |
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2. Transfer the separated proteins onto an Immobilon P membrane (Millipore)
and incubate the membrane for 1 hour at room temperature or overnight at 2-8 °C in Blocking
Solution (1 X PBS, pH 7.4 containing 5% dry milk). | |
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3. Wash the membrane at room temperature for 30 minutes with 5 changes of Wash Buffer (1X PBS with 0.1% NP40,). | |
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4. Incubate the membrane for 2 hours at room temperature or overnight at 2-8 °C
in Blotting Buffer (1 X PBS, pH 7.4 containing 5% dry milk unless otherwise indicated) containing primary
antibody ( 0.2 to 1ug/ml concentration as indicated on datasheet). | |
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5. Wash the membrane at room temperature for 30 minutes with 5 changes of Wash Buffer. | |
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6. Incubate the membrane at room temperature for 1 hour in Blotting Buffer
containing an appropriate secondary reagent such as goat anti rabbit IgG or anti-mouse IgG -HRP at
1:10,000 (Catalog # AviHRP-GAR ). | |
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7. Wash the membrane at room temperature for 30 minutes with 5 changes of Wash Buffer. | |
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8. Detect with chemiluminescence reagents. (Catalog # Cat. No. AviLight-500). | |
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9. Optimal dilutions should be determined by each laboratory for each application. | |
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