|H Mk||Endogenous||53||Rabbit IgG|
Western blot analysis of extracts from various cell lines using Rad23B (D4W7F) Rabbit mAb.Learn more about how we get our images.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human RAD23A protein (hRAD23A-Myc/DDK; +) and Myc-tagged full-length human RAD23B protein (hRAD23B-Myc; +), using Rad23B (D4W7F) 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 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.
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 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 October 2016
Protocol Id: 10
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.
Rad23B (D4W7F) Rabbit mAb recognizes endogenous levels of total Rad23B protein. This antibody does not cross-react with Rad23A protein.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala140 of human Rad23B protein.
The yeast nucleotide excision repair (NER) radiation sensitive protein 23 (rad23) and its human homologs Rad23A (hHR23A) and Rad23B (hHR23B) are critical components of the cellular machinery that recognize DNA lesions and serve as receptors that target ubiquitinated substrates to the proteasome for degradation (1).
The UV excision repair protein Rad23B is a multi-domain scaffold protein that plays an important role in ubiquitin-dependent proteasomal degradation. Rad23B contains an amino-terminal ubiquitin-like (UbL) domain that facilitates interaction with the S5a/PSMD4 subunit of the proteasome 19S regulatory complex (2,3). In addition, Rad23B contains a central ubiquitin-associated domain (UBA1) and a carboxy-terminal UBA2 domain, which bind mono- and polyubiquitin with distinct specificities (4). Research studies demonstrate that Rad23B binds specifically to K48-ubiquitinated proteins to facilitate recruitment of target proteins to the proteasome (5). Between the paired UBA domains, Rad23B contains an XPC-binding domain that facilitates binding to XPC and recruitment to DNA lesions (6), as well as the binding of peptide:N-glycanase that is critical for recruitment of ubiquitinated ERAD substrates to the proteasome (7). Research studies have shown that targeted deletion of the murine Rad23b locus impairs embryonic development, suggesting that Rad23B is essential for mammalian development (8).
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