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83732
Poly/Mono-ADP Ribose (E6F6A) Rabbit mAb
Primary Antibodies

Poly/Mono-ADP Ribose (E6F6A) Rabbit mAb #83732

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R All Endogenous Rabbit IgG
Western Blotting

Western blot analysis of Colo 205 cells treated (+) with combinations of the following treatments as indicated: hydrogen peroxide (500 μM, 5 min), hydrogen peroxide-treated lysates treated with phosphodiesterase 1 (0.5 μg/mL, 4 hr at 37ºC), or with tcPARG (5 μM, 4 hr at 37ºC ) using Poly/Mono-ADP Ribose (E6F6A) Rabbit mAb (upper), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

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Western Blotting

Western blot analysis of Colo 205 cells untreated (-), or treated with hydrogen peroxide (500 μM, 5 min; +), using Poly/Mono-ADP Ribose (E6F6A) Rabbit mAb (upper), or β-Tubulin (9F3) Rabbit mAb #2128 (lower).

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.

A. Solutions and Reagents

From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product datasheet) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised November 2013

Western Blot Reprobing Protocol

Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Wash Buffer: Tris Buffered Saline with Tween® 20 (TBST-10X) (#9997)
  2. Stripping Buffer: To prepare 100 ml, mix 6.25 ml of 1M Tris-HCl pH 6.8, 10 ml of 20% SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H20. Make buffer fresh just prior to use.

B. Protocol

  1. After film exposure, wash membrane four times for 5 min each in TBST. Best results are obtained if the membrane is not allowed to dry.
  2. Incubate membrane for 30 min at 50°C in stripping buffer (with slight agitation).
  3. Wash membrane six times for 5 min each in TBST.
  4. (Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.
  5. Wash membrane again four times for 5 min each in TBST.
  6. The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

posted June 2005

revised October 2016

Protocol Id: 10

Application Dilutions
Western Blotting 1:1000
Storage:

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Poly/Mono-ADP Ribose (E6F6A) RmAb recognizes endogenous levels of ADP ribosylated proteins and does not cross-react with other post translational modifications.

Species Reactivity:

Human, Mouse, Rat, All Species Expected

Monoclonal antibody is produced by immunizing animals with KLH modified on lysines with ADP ribose.

ADP-ribosylation is a post-translational modification that has been described to occur on the side chain of several acceptor residues (lysine, arginine, glutamate, aspartate, cysteine, serine) and protein amino termini as well as on DNA and tRNA (1). ADP-ribosyl transferases (ADPRTs) catalyze the transfer of ADP-ribose from β-NAD+ and release nicotinamide in the process. Mono-ADP-ribosyl transferases (MARTs, or monoPARPs) comprise the vast majority of the ADPRTs. These monoenzymes, which include the sirtuins and many of the PARP (ARTD) and ART proteins, transfer a single ADP-ribose unit to the target residue (MARylation). The poly-ADP-ribose polymerases (polyPARPs) or polyenzymes, which include human PARP1, 2, 5a and 5b, are the most widely studied and can polymerize linear or branched chains of up to ~200 ADPR units (2). Specificity is determined primarily, but not exclusively, by a nonconsecutive catalytic triad motif, with some exceptions. Those containing the R-S-E motif like Cholera toxin are arginine-directed transferases, while those containing the H-Y-E triad tend to exhibit polymerase activity (3,4). ADP-ribosylation is reversible and can be degraded down to a single ADP-ribose unit by poly-ADP-ribose glycohydrolase (PARG) or ADP-ribosylhydrolase 3 (ARH3) or completely removed from the target residue by ARH1, TARG1, MacroD1 or MacroD2 (5).

ADP-ribosylation is involved in a variety of cellular processes, including mitotic spindle formation, chromatin decondensation, cell stress response, retroviral silencing, RNA biology, and transcription, but the most well-known function of ADP-ribose chains is to serve as a scaffold for recruiting DNA repair proteins that contain PAR-binding modules to sites of DNA damage (6). X-ray repair cross-complementing protein 1 (XRCC1), histone macroH2A1, RNF146 (Iduna) an E3 ubiquitin ligase, and many of the PARPs themselves, among others, contain PAR-binding motifs (PBMs) or domains: WWE, PAR-binding zinc-finger (PBZ), or macrodomains (7). PARylation has a central role in cell survival, and is tightly regulated. PARP deficiency can leave a cell vulnerable to DNA damage-induced apoptosis, while hyper PARylation can lead to parthanatos, a unique form of cell death (8). The role of PARylation in DNA repair has inspired great interest in developing candidate drug inhibitors for PARP, in particular to treat breast, prostate and small cell lung cancers with mutations in DNA repair genes like BRCA1/2, CHK2 or ATM. Stat1, PERK, p53, G-actin and Ras are just a few examples of proteins that are functionally modulated by ADP-ribosylation (6,7). Modification by ADP-ribose can block protein interactions or, in the case of P2X7, cause a conformational change that in the presence of ART2 expression sensitizes naive murine T-cells to extracellular NAD+ leading to apoptosis (9).

  1. Koch-Nolte, F. et al. (2008) Front Biosci 13, 6716-29.
  2. Leung, A.K. (2014) J Cell Biol 205, 613-9.
  3. Laing, S. et al. (2011) Amino Acids 41, 257-69.
  4. Vyas, S. et al. (2014) Nat Commun 5, 4426.
  5. Vivelo, C.A. and Leung, A.K. (2015) Proteomics 15, 203-17.
  6. Gupte, R. et al. (2017) Genes Dev 31, 101-126.
  7. Wei, H. and Yu, X. (2016) Genomics Proteomics Bioinformatics 14, 131-139.
  8. David, K.K. et al. (2009) Front Biosci (Landmark Ed) 14, 1116-28.
  9. Seman, M. et al. (2003) Immunity 19, 571-82.
For Research Use Only. Not For Use In Diagnostic Procedures.

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Product # Size Price
83732S
100 µl (10 western blots) N/A

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