Editor's Note: All antibody reviews appear in protocol format.
Primary Antibody: Cleaved PARP Antibody
Antigen/Epitope: Synthetic peptide (KLH coupled) corresponding to carboxy-terminal residues surrounding Asp214 in human PARP
Supplier and catalog number: Cell Signaling Technology 9541
Sample: Whole cell lysate from cultured A549 (a non small cell lung cancer cell line) cells, 20 ug total protein (as determined by Bradford)
Secondary Antibody: Goat anti-rabbit IgG-HRP
Supplier and catalog number: Santa Cruz Biotechnology SC2004
Block: 5% nonfat milk in TBS
Incubation time: 90 min
Incubation temperature: 4°C
Primary Antibody Incubation
Dilution used: 1/1000
Diluent composition: TBS
Incubation time: overnight
Incubation temperature: 4°C
Secondary Antibody Incubation
Dilution used: 1/10,000
Diluent composition: TBS
Incubation time: 1 hr
Incubation temperature: room temperature
Detection Method: SuperSignal West Pico Chemiluminescent Substrate from Thermo Scientific Pierce Protein Research Products. I generally do a 2 min exposure and detect on film.
Results Summary:I've run this experiment several times with primary Ab dilutions ranging from 1:100 to 1:2000. I have found that 1:1000 gives a very good signlal to noise when performing an overnight incubation. Good signal; no non-specific bands coming up.
Additional notes/tips: I blot onto PVDF membrane.
(Note: The image at the top of this page is from Cell Signaling Technology.)
Assistant Research Faculty
Department of Medicine, David Geffen School of Medicine
University of California, Los Angeles