Cleaved PARP Antibody from Cell Signaling Technology, used in Western Blotting

Cleaved PARP Antibody from Cell Signaling Technology, used in Western Blotting
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
Cleaved PARP Antibody from Cell Signaling Technology, used in Western Blotting
The Good

Good signal; no non-specific bands coming up.

The Bad

None.

The Bottom Line

Works well.

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