このprotocolでやってみてください。ただし10 cm dishでは細胞が多すぎると思います。Actinならば6-well(あるいは35 mm dish)でも検出できるはずです。それから、Blocking solutionは、必ず、用事調整してください。感度が顕著に下がることがあります。
1. (Optional) Wash cells with icne-cold PBS (-) twice.
2. Harvest cells in 1 ml PBS (-) using Cell Lifter (Corning) on ice and transfer the suspension to a 1.5 ml microsentrifuge tube (an aliquot can be kept for protein assay.) So far, Cell Lifter is the best to harvest cells from a plastic dish.
3. Spin at 5,000 rpm for 5 min at 4C.
4. Remove sup.
5. Add 100 μl of 1X SDS sample buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 62.5 mM Tris-HCl, pH6.8) to the pellet (if the pellet is bigger than 10-20 μl, use 2X sample buffer to make the final concentration of the sample buffer to 1X, e.g. add 100 μl of 2X sample buffer and 70 μl of distilled water to a 30 μl cell pellet).
6. Resuspend the pellet until it becomes fluid (until the viscosity becomes negligible for pipetting.)
7. (Optional) Boil for 3 min.
8. Apply 10 to 20 μl/lane to a SDS gel (it would be a good idea to use a prestained molecular weight marker such as Rainbow Marker from GE HealthCare, formerly Amersham, with your samples.)
9. After transfer, stain the membrane (PVDF or nitrocellulose) with 0.1% Ponceau S in 5% acetic acid (wash the membrane with distilled water 3 times and stain with the Ponceau solution for 5 min. Destain with distilled water. Protein bands should be visible immediately. It won't destain completely, just block with dried milk which will knock off the dye.)
10. Follow normal Western blot procedures, if you see nice protein bands..... |
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