discontinued

BP7143 PARP-1 (cleaved) antibody

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0.1 ml / €430.00

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Rabbit anti Human, Mouse, Rat PARP-1 (cleaved)

BP7143

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  • BP7143

Product Description for PARP-1 (cleaved)

Rabbit anti Human, Mouse, Rat PARP-1 (cleaved).
Presentation: Aff - Purified
Product is tested for Immunocytochemistry/Immunofluorescence, Western blot / Immunoblot.

Properties for PARP-1 (cleaved)

Product Category Primary Antibodies
Quantity 0.1 ml
Synonyms ADP-ribose, ADPRT, ADPRT1, PARP, PARP-1, PARP1 - poly polymerase family, PPOL, member 1, pADPRT-1
Presentation Aff - Purified
Reactivity Hu, Ms, Rt
Applications ICC/IF, WB
Clonality Polyclonal
Host Rabbit
Shipping to Worldwide
PDF datasheet View Datasheet
Manufacturer Acris Antibodies GmbH
Material safety datasheet MSDS for Polyclonal Antibodies (de)

Datasheet Extract

Immunogen
Immunogen:
Chemically synthesized peptide corresponding to the N-terminus of cleavage site (214/215) of human PARP.
Application Western blot (1:1000).
Positive Controls Used: Jurkat or HeLa cells treated with staurosporine or etoposide (25 µM for 3 hours).
Previous lots have been used in immunostaining applications.
Background Poly (ADP-Ribose) Polymerase (PARP) is a 116 kDa nuclear protein which is strongly activated by binding to DNA strand breaks. PARP plays a role in DNA repair as well as in other cellular processes, including DNA replication, cell proliferation and differentiation. During apoptosis, ICE family members, such as caspase-3 and -7, cleave PARP to yield an 85 kDa and a 25 kDa fragment. PARP cleavage is considered to be one of the classical characteristics of apoptosis. This antibody specifically recognizes the 85 kDa fragment of cleaved PARP and can be used as a marker for detecting apoptotic cells.
Protocols Western Blotting Procedure

1. Lyse approximately 10e7 cells in 0.5 mL of ice cold Cell Lysis Buffer (formulation provided below). This buffer, a modified RIPA buffer, is suitable for recovery of most proteins, including membrane receptors, cytoskeletal-associated proteins, and soluble proteins. Other cell lysis buffer formulations, such as Laemmli sample buffer and Triton-X 100 buffer, are also compatible with this procedure. Additional optimization of the cell stimulation protocol and cell lysis procedure may be required for each specific application.
2. Remove the cellular debris by centrifuging the lysates at 14,000 x g for 10 minutes. Alternatively, lysates may be ultracentrifugedat 100,000 x g for 30 minutes for greater clarification.
3. Carefully decant the clarified cell lysates into clean tubes and determine the protein concentration using a suitable method, such as the Bradford assay. Polypropylene tubes are recommended for storing cell lysates.
4. React an aliquot of the lysate with an equal volume of 2x Laemmli Sample Buffer (125 mM Tris, pH 6.8, 10% glycerol, 10% SDS, 0.006% bromophenol blue, and 130 mM dithiothreitol [DTT]) and boil the mixture for 90 seconds at 100°C.
5. Load 10-30 µg of the cell lysate into the wells of an appropriate single percentage or gradient minigel and resolve the proteins by SDS-PAGE.
6. In preparation for the Western transfer, cut a piece of PVDF membrane slightly larger than the gel. Soak the membrane in methanol for 1 minute, then rinse with ddH2O for 5 minutes. Alternatively, nitrocellulose may be used.
7. Soak the membrane, 2 pieces of Whatman paper, and Western apparatus sponges in transfer buffer (formulation provided below) for 2 minutes.
8. Assemble the gel and membrane into the sandwich apparatus.
9. Transfer the proteins at 140 mA for 60-90 minutes at room temperature.
10. Following the transfer, rinse the membrane with Tris buffered saline for 2 minutes.
11. Block the membrane with blocking buffer (formulation provided below) for one hour at room temperature or overnight at 4°C.
12. Incubate the blocked blot with primary antibody at a 1:1000 starting dilution in Tris buffered saline supplemented with 3% Ig-free BSA and 0.1% Tween 20 overnight at 4°C or for two hours at room temperature.
13. Wash the blot with several changes of Tris buffered saline supplemented with 0.1% Tween 20.
14. Detect the antibody band using an appropriate secondary antibody, such as goat F(ab)2 anti-rabbit IgG alkaline phosphatase conjugate or goat F(ab)2 anti-rabbit IgG horseradish peroxidase conjugate in conjunction with your chemiluminescence reagents and instrumentation.

Cell Lysis Buffer Formulation:
10 mM Tris, pH 7.4
100 mM NaCl
1 mM EDTA
1 mM EGTA
1 mM NaF
20 mM Na4P2O7
2 mM Na3VO4
0.1% SDS
0.5% sodium deoxycholate
1% Triton-X 100
10% glycerol
1 mM PMSF (made from a 0.3 M stock in DMSO)
or 1 mM AEBSF (water soluble version of PMSF)
60 µg/mL aprotinin
10 µg/mL leupeptin
1 µg/mL pepstatin
(alternatively, protease inhibitor cocktail such as Sigma Cat. # P2714 may be used)



Transfer Buffer Formulation:
2.4 gm Tris base
14.2 gm glycine
200 mL methanol
Q.S. to 1 liter, then add 1 mL 10% SDS.
Cool to 4°C prior to use.

Tris Buffered Saline Formulation:
20 mM Tris-HCl, pH 7.4
0.9% NaCl

Blocking Buffer Formulation:
100 mL Tris buffered saline
5 gm BSA
0.1 mL Tween 20
General Readings Le Page, F., et al. (2003) Poly(ADP-ribose) polymerase-1 (PARP-1) is required in murine cell lines for base excision repair of oxidative DNA damage in the absence of DNA polymerase beta. J. Biol. Chem. 278(20):18471-18477.
Turturro, F., et al. (2002) Model of inhibition of the NPM-ALK kinase activity by herbimycin A. Clin. Cancer Res. 8(1):240-245 (cites the use of this antibody).
Leemans, J.C., et al. (2001) Depletion of alveolar macrophages exerts protective effects in pulmonary tuberculosis in mice. J. Immunol. 166(7):4604-4611 (cites the use of this antibody).
Soldani, C. and A.I. Scovassi (2002) Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update. Apoptosis 7(4):321-328.
Germain, M., et al. (1999) Cleavage of automodified Poly (ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7. J. Biol. Chem. 274(40):28379-28384.
Kaufmann, S.H., et al. (1993) Specific proteolytic cleavage of poly (ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res. 53(17):3976-3985.
Storage Store the antibody at -20 °C. Can be shipped at 2 - 8 °C.
Avoid repeated freezing and thawing.
Centrifuge vial before opening.
Shelf life: One year from despatch.
Format
Purification:
Affinity chromatography using a peptide corresponding to the PARP cleavage site.
Buffer System:
Dulbecco's phosphate buffered saline (without Mg2+ and Ca2+), pH 7.3
(+/- 0.1), 50% glycerol with 1.0 mg/mL BSA (IgG, protease free) as a carrier, containing
0.05 % sodium azide as preservative
State:
Liquid Ig fraction
Aff - Purified
Specificity
Specificity:
This antibody detects PARP.
Species:
Human, Mouse, Rat.

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