|Immunofluorescence (Immunocytochemistry)||1:12000 - 1:48000|
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 product webpage for recommended antibody dilution.
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.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 10
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted December 2010
Protocol Id: 3
Human, 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).
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