53BP1 (P550) Antibody 53BP1 (P550)抗体

Applications Key:  W=Western Blotting
Reactivity Key:  H=Human  Mk=Monkey
Species cross-reactivity is determined by Western blot.

Protocols

4908:

Western Blotting

Specificity / Sensitivity

53BP1 (P550) Antibody detects endogenous levels of total 53BP1 protein.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser552 of human 53BP1.

Western Blotting

Western blot analysis of extracts from various cell types using 53BP1 (P550) Antibody.

53BP1 (P550) Antibody 53BP1 (P550)抗体

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.