Phospho-53BP1 (Ser1778) Antibody 磷酸53BP1（Ser1778）抗体

Applications Key:  W=Western Blotting  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  Mk=Monkey
Species cross-reactivity is determined by Western blot.

Protocols

2675:

Flow, Immunofluorescence, Western Blotting

Specificity / Sensitivity

Phospho-53BP1 (Ser1778) Antibody detects endogenous levels of 53BP1 only when phosphorylated at serine 1778.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser1778 of human 53BP1. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western blot analysis of extracts from 293 cells, untreated or UV-treated (50 mJ for 2 hours), using Phospho-53BP1 (Ser1778) Antibody (upper) or 53BP1 Antibody #4937 (lower).

Flow Cytometry

Flow cytometric analysis of HeLa cells, untreated (blue) or UV-treated (green), using Phospho-53BP1 (Ser1778) Antibody compared with a nonspecific negative control antibody (red).

IF-IC

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or UV-treated (right), using Phospho-53BP1 (Ser1778) Antibody (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

 

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.