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 and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
Rad50 antibody detects endogenous levels of total Rad50 protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Lys713 of human Rad50. Antibodies are purified by protein A and peptide affinity chromatography.
The DNA repair protein Rad50 is a member of the structural maintenance of chromosomes family (SMC) and plays an important role in cell cycle checkpoint signaling and double-strand break repair in response to DNA damage (1-4). Rad50 forms a complex with Mre11 and Nbs1 that becomes activated in response to DNA damage (3). In normal human cells, the MRN complex acts to tether linear DNA molecules, providing a flexible link between DNA ends (1). Genomic instability and cancer have been shown to develop in cells with genetic mutations affecting the proteins in the MRN complex (2). ATM-dependent phosphorylation of Rad50 at Ser635 in response to DNA damage is important in regulating downstream signaling, DNA repair and checkpoint control (5).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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