Nonphospho-4E-BP1 (Thr46) (87D12) Rabbit mAb 兔单克隆抗体

Applications Key:  W=Western Blotting  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
Species cross-reactivity is determined by Western blot.

Protocols

4923:

Flow, Western Blotting

Specificity / Sensitivity

Nonphospho-4E-BP1 (Thr46) (87D12) Rabbit mAb detects endogenous levels of 4E-BP1 only when dephosphorylated at Thr46. The antibody cross-reacts with 4E-BP2 and 4E-BP3 dephosphorylated at equivalent sites.

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Thr46 of human 4E-BP1.  

Western Blotting

Western blot analysis of extracts from COS cells, treated with λ phosphatase or 20% FBS as indicated, using Nonphospho-4E-BP1 (Thr46) (87D12) Rabbit mAb (upper), Phospho-4E-BP1 (Thr37/46) Antibody #9459 (middle), and 4E-BP1 Antibody #9452 (lower).
Nonphospho-4E-BP1 (Thr46) (87D12) Rabbit mAb 兔单克隆抗体

For Western blots, incubate membrane with diluted antibody in 5% w/v BSA, 1X TBS, 0.1% Tween-20 at 4°C with gentle shaking, overnight.

Products available from Cell Signaling Technology are linked by their respective catalog numbers.

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.