Western blot analysis of extracts from various cell lines using Rad9A (D2J4P) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).Learn more about how we get our images.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human Rad9A (Rad9A-Myc/DDK; +) protein, using Rad9A (D2J4P) Rabbit mAb (upper) or Myc-Tag (71D10) Rabbit mAb #2278 (lower).Learn more about how we get our images.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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.
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 November 2013
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.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
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.
Rad9A (D2J4P) Rabbit mAb recognizes endogenous levels of total Rad9A protein. Based on the amino acid sequence of the immunogenic peptide, this antibody is not expected to cross-react with Rad9B protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro31 of human Rad9A protein.
DNA damage resulting from genotoxic stress activates cellular checkpoints that prevent or delay cell division until either damaged DNA is repaired or the cell follows an apoptotic pathway. The Rad9 homolog A (Rad9A, Rad9) protein is part of a checkpoint protein complex that acts as an early sensor of DNA damage. Together with the Hus1 and Rad1 checkpoint proteins, Rad9 forms a heterotrimeric 9-1-1 complex with a ring structure similar to the processivity factor PCNA. The 9-1-1 complex induces multiple signaling pathways, including the ATM and ATR-activated DNA repair pathways (1,2). A functional 9-1-1 complex is required for ATR-dependent S phase checkpoint signaling (3).
The 9-1-1 complex interacts with DNA topoisomerase 2-binding protein 1 (TopBP1) in response to DNA damage, activating ATR and causing signal amplification through further recruitment of TopBP1 (4). The 9-1-1 complex interacts with DNA mismatch repair proteins MSH2, MSH3, and MSH6 to play a role in mismatch repair (5). During an error-free DNA damage tolerance process, the 9-1-1 complex cooperates with polyubiquitinated PCNA and Exo1 nuclease to support switching of the replicative polymerase to the undamaged template (6).
Research studies indicate that the two Rad9 paralogues (Rad9A and Rad9B) can both functionally complement one another and display distinct biological functions.
Specifically, Rad9B senses nucleolar stress and causes a delay in the cell cycle at G1/S phase (7).
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|14484S||100 µl (10 western blots)||$ 255.0|