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Index / Trevigen / Anti_PARP Monoclonal Antibody (Clone C2_10) /Product Detail : 4338-MC-50 Anti_PARP Monoclonal Antibody (Clone C2_10)
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#4338-MC-50 Anti_PARP Monoclonal Antibody (Clone C2_10)

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  Price : 349   EUR
397   USD
271   GBP
1469   Zloty
46806   JPY
2697   NOK
2890   SEK
395   CHF

Product name : Anti_PARP Monoclonal Antibody (Clone C2_10)

Catalog number : 4338-MC-50

Quantity: 50 ug

Availability: Yes

Supplier name : Trevigen

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About this Product :

Anti_PARP Monoclonal Antibody (Clone C2_10) monclonal andibody monoclonal antobodies are directed against a specific epitope. The use Anti_PARP Monoclonal Antibody (Clone C2_10) is much more reproducable than with a polyclonal antibody.
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Anti_PARP Monoclonal Antibody (Clone C2_10) http://biotech-map.com/aa_search/table_sample.html?q=Mab&filter=
Advisory
To keep the quality and the affinity of the antibodies cycles of freezing and thawing should be avoided. For antibodies in a liquid form or reconstituted lyophilized antibodies small amounts of the soultion could be captured on the cap or the walls of the container. Right before use you could briefly centrifuge the vial to collect all of the solution on the bottom.

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Anti-PARP Monoclonal Antibody 

Description: Poly(ADP-ribose) polymerase 1 (PARP-1) is a 114 kDa eukaryotic enzyme, which uses NAD as a substrate to form poly(ADP-ribose) (PAR cat# 4336-100-01). Poly(ADPribosyl) ation of proteins occurs by the addition of an ADP-ribose (generated from NAD+) to glutamic acid residues. PARP-1 ribosylates itself at an internal automodification domain. Self modification causes PARP-1 to lose enzymatic activity and dissociate from DNA. PARP-1 enzymatic activity is restored by poly(ADP-ribose) glycohydrolase, which degrades PAR into monomers. During apoptosis, PARP-1 activity initially increases, but later, falls due to automodification and cleavage by caspases. The specific rate of change in PARP-1 activity is dependent upon a variety of factors including cell type, method of induction of DNA damage or apoptosis, and culture conditions. Specific proteolytic cleavage of PARP has been demonstrated to be a reliable marker for apoptosis in a wide variety of cell types, generating 85 kDa and 26 kDa fragments. Trevigen’s C2-10 antibody recognizes full length PARP and the 85 KDa cleavage product, which contains the catalytic, automodification and NAD-binding domains.
Physical State: C2-10 is an IgG1 and is provided in 1X PBS/0.1 mg/ml BSA/0.01% sodium azide.
Immunogen: Calf Thymus PARP-1.
Specificity: Mouse monoclonal antibody C2-10 detects the 114 kDa PARP-1 holoenzyme, 85 kDa apoptosis-related, and necrosis-related 50 kDa, 62 kDa and 74 kDa cleavage fragments.
It cross-reacts with human, monkey, hamster, rat and mouse PARP-1. It does not recognize the chicken equivalent.
Storage: Store at 4°C for at least 1 month, or store aliquots at -20°C.

Cell Lysates for Western Blotting:
II. Preparation of Cell Extracts for SDS-PAGE
Choose one of the following methods for the preparation of cell extracts for Western blotting application. Two methods are provided. The purpose of urea in the sample buffer and sonication step is to effectively dissociate PARP:DNA interactions. Alternatively, you may want to use a non-denaturing procedure:
A. Phosphate buffer extraction method (non-denaturing):
The protocol given below is for adherent cells. For suspension cells, harvest cells by centrifugation, and resuspend the cell pellet in each buffer by tapping the tube gently.
1. For best results use around 1-2 x 107 cells.
2. Chill plates or dishes on ice.
3. Aspirate medium.
4. Rinse in ice cold Buffer A.
5. Cover cells with 5 ml of cold Buffer B, let stand on ice for 20 min. Drain buffer.
6. Cover cells with 2 ml of cold Buffer C, let stand on ice for 20 min.
7. Recover Buffer C-cell mixture and transfer to a microcentrifuge tube prechilled on ice. Centrifuge at 3,000 x g for 10 min at 4°C. Recover supernatant to prechilled tube and place on ice.
8. Optional: to increase protein recovery, add 1 ml ice cold Buffer C to the cell pellet and vortex to resuspend. Leave on ice for 10 minutes. Centrifuge at 3000 x g for 10 min at 4°C. Recover supernatant and combine with sample recovered in step 7.
9. Store the cell extract at -20°C until analysis.
10. Add 2 volumes of urea sample buffer and subject sample to SDS-PAGE and Western blot analysis.
B. Sonication/Urea extraction method
1. If necessary, scrape cells from dish or plate in a small volume ice cold 1X PBS.
2. Harvest cells by centrifugation at 500 x g for 5 minutes at 4 °C.
3. Wash cell pellet in ice cold 1X PBS.
4. Resuspend cells at 107 cells/ml in Sample Buffer. Use a 1000 μl pipette to resuspend the cells. The sample will be viscous due to release of DNA during lysis.
5. Sonicate sample on ice for 15 seconds, and incubate at 65 °C for 15 minutes.
6. Store sample at -20°C until analysis.
7. Add 2 volumes of sample buffer and use in SDS-PAGE and Western analysis.

III. SDS-PAGE and Western Analysis
Methods for SDS-PAGE and Western transfer are available in a wide variety of techniques manuals. For Western blotting a 1:2000 dilution using enhanced chemiluminescence (ECL) or 1:1000 using alkaline phosphatase should be used but titration may be needed for optimal results.

1. Heat prepared cell extract at 95°C for 10 minutes.
2. Load sample (ideally, 20 - 40 μg total protein) onto 10% or 12% SDS polyacrylamide
gel. Include prestained markers for reference. Run SDS-PAGE.
3. Transfer proteins to nitrocellulose by Western blot.
4. Block membrane with 1X PBS, 5% non-fat dried milk, 0.1% Tween® 20; 150 mM NaCl, for 1 hr at room temperature, with gentle agitation.
5. Incubate membrane with a 1:2000 dilution of Anti-PARP (C2-10) in 1X PBS, 5%
non-fat dried milk, 0.1% Tween® 20; 150 mM NaCl, for 1 hour at room temperature or overnight at 4°C.
6. Wash membrane three times with 1X PBS, 0.1% Tween® 20, for 10 minutes per wash.
7. Incubate membrane with secondary antibody for 1 hr at room temperature (use dilution recommended by antibody manufacturer).
8. Wash membrane three times with 1X PBS, 0.1% Tween® 20, for 10 minutes per wash.
9. Incubate with appropriate color development or chemiluminescent reagents according to manufacturer’s instructions.
Note: If a phosphatase conjugated secondary antibody is used, the 1X PBS should be replaced with 50 mM Tris-Cl, pH 7.4, 150 mM NaCl.

IV. Immunocytochemistry
C2-10 can also be used in immunocytochemistry applications. The recommended antibody dilution is 1:1000 but for optimal results the antibody should be titrated. Note that the antibody will recognize the full length and 85 KDa cleaved fragment generated during apoptosis.

 

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