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我这有一个关于磷酸化抗体的推荐步骤：其中讲到最好不要煮。
1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on either a bovine
cardiac preparation prepared as described in the technical note below or an
experimental sample; transfer the proteins to nitrocellulose. Do not boil
preparation. Warm to 37°C for 30-45 minutes before loading the sample.
2. Wash the blotted nitrocellulose twice with water.
3. Block the blotted nitrocellulose in freshly prepared TBS containing 5% nonfat dry milk and 0.05% Tween 20 (TBST-MLK) for 20 minutes at room temperature with constant agitation.
4. Incubate the nitrocellulose with 0.5-2μg/ml of anti-phospho-Phospholamban
(Ser16), diluted in freshly prepared TBS-MLK overnight with agitation at 4°C.
5. Wash the nitrocellulose twice with water.
6. Incubate the nitrocellulose in the secondary reagent of choice (a goat anti-rabbit HRP conjugated IgG, Catalog # 12-348, 1:5000 dilution was used) in TBS-MLK for 1.5 hours at room temperature with agitation.
7. Wash the nitrocellulose twice with water.
8. Wash the nitrocellulose in TBS-0.05% Tween 20 for 10 minutes.
9. Rinse the nitrocellulose in 4-5 changes of water.
10. Use detection method of choice (enhanced chemiluminescence was used).
