Western Blot/Anti-P-CREB
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Treat cells. For PC12 or NIH 3T3 cells a moderately confluent 60 mm plate is used.
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Aspirate media. Rinse cells with PBS, aspirate.
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Lyse cells in 200 uL of SDS sample buffer (100 oC). Immediately scrape the cells off the plate with a rubber policeman.
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Put the extract into an eppendorf and boil for 5 min., cool on ice.
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Load onto 10% SDS-PAGE. I usually load about 1/5 of the lysate from a 60 mm dish. This is about 100 ug protein for PC12 cells.
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After electrophoresis, electrotransfer to nitrocellulose membrane. I include in the SDS-PAGE prestained molecular weight markers to check the efficiency of the transfer and for M.W. determination.
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After transfer, rinse the blot 2 times with TBST (10 mM Tris (pH 7.4), 150 mM NaCl, 0.05% Tween-20).
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Block with 4% BSA in TBST for 45 - 60 minutes. I use RIA grade BSA from USB (United States Biochemical; Catalog number 10868). I also include 0.02% NaN3 in this block and reuse it at least 5 times (Keep in 4oC between uses). We have had problems obtaining good signals when we've used poor quality BSA. Others have told me that 10% nonfat dry milk works well for anti-PCREB, but in my hands milk is not as good as BSA from USB (it seems to mask the epitope).
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Remove block solution and add primary antibody. For antiphosphoCREB (IgG, 0.7 mg/ml), I use 1:5,000 - 1:20,000 dilution in 4% BSA in TBST. This should be titrated for optimal results. I incubate with primary Ab 3 hours - overnight at room temp with shaking.
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Remove the antibody solution and wash the blot 3 times, 5-7 minutes each, with 0.5% BSA in TBST.
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Incubate with Goat anti-Rabbit/alkaline phosphatase (from Promega; 1:7500 inTBST with 5% BSA) for 1 hour at room temp with shaking. I have also used successfully Amersham ECL detection which employs goat anti-Rabbit HRP.
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Wash filter 3 times 7 minutes with 0.5% BSA in TBST.
- Perform Alkaline phosphatase reaction using NBT and BCIP substrates. I find that CREB usually comes up within several minutes of adding the AP substrates, however a one hour reaction time may be necessary.
