Phospho-4E-BP1 (Ser65) Antibody 磷酸- 4E - BP1（Ser65）抗体

Western Blotting

Western blot analysis of extracts from 293 cells using 4E-BP1 Antibody #9644 (lower) and Phospho-4E-BP1 (Ser65) Antibody #9451 (upper). The cells were starved for 24 hours in serum-free medium and underwent a 1 hour amino acid deprivation. Amino acids were replenished for 1 hour. Cells were then either untreated (-) or treated with 100 nM insulin (+)
 

A. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

1. 1X Phosphate Buffered Saline (PBS).
2. 1X SDS Sample Buffer: (#7722, #7723) 62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue or phenol red.
3. Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol (pH 8.5).
4. 10X Tris Buffered Saline (TBS): (#9997) To prepare 1 liter of 10X TBS: 24.2 g Tris base, 80 g NaCl; adjust pH to 7.6 with HCl (use at 1X).
5. Nonfat Dry Milk: (#9999) (weight to volume [w/v]).
6. Blocking Buffer: 1X TBS, 0.1% Tween-20 with 5% w/v nonfat dry milk; for 150 ml, add 15 ml 10X TBS to 135 ml water, mix. Add 7.5 g nonfat dry milk and mix well. While stirring, add 0.15 ml Tween-20 (100%).
7. Wash Buffer: 1X TBS, 0.1% Tween-20 (TBS/T).
8. Bovine Serum Albumin (BSA): (#9998).
9. Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween-20 with 5% BSA; for 20 ml, add 2 ml 10X TBS to 18 ml water, mix. Add 1.0 g BSA and mix well. While stirring, add 20 μl Tween-20 (100%).
10. Phototope^®-HRP Western Blot Detection System: (#7071 anti-rabbit) or (#7072 anti-mouse) Includes biotinylated protein ladder, secondary (#7074 anti-rabbit) or (#7076 anti-mouse) antibody conjugated to horseradish peroxidase (HRP), anti-biotin antibody conjugated to HRP, LumiGLO^® chemiluminescent reagent and peroxide.
11. Prestained Protein Marker, Broad Range (Premixed Format): (#7720).
12. Biotinylated Protein Ladder Detection Pack: (#7727).
13. Blotting Membrane: This protocol has been optimized for nitrocellulose membranes, which CST recommends. PVDF membranes may also be used.

B. Protein Blotting

A general protocol for sample preparation is described below.

1. Treat cells by adding fresh media containing regulator for desired time.
2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
3. Lyse cells by adding 1X SDS sample buffer (100 μl per well of 6-well plate or 500 μl per plate of 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
4. Sonicate for 10–15 seconds to shear DNA and reduce sample viscosity.
5. Heat a 20 μl sample to 95–100°C for 5 minutes; cool on ice.
6. Microcentrifuge for 5 minutes.
7. Load 20 μl onto SDS-PAGE gel (10 cm x 10 cm). NOTE: CST recommends loading prestained molecular weight markers (#7720, 10 μl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 μl/lane) to determine molecular weights.
8. Electrotransfer to nitrocellulose or PVDF membrane.

Phospho-4E-BP1 (Ser65) Antibody 磷酸- 4E - BP1（Ser65）抗体