Western blot analysis of extracts from various cell lines using HELQ (D4K2O) Rabbit mAb.
Western blot analysis of extracts from wild type or HELQ-knockout U-2 OS cells using HELQ (D4K2O) Rabbit mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb #64873 (lower).
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing DDK-tagged full-length human HELQ (hHELQ-DDK; +), using HELQ (D4K2O) Rabbit mAb (upper) or DYKDDDDK Tag (D6W5B) Rabbit mAb #14793 (lower).
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.
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
HELQ (D4K2O) Rabbit mAb recognizes endogenous levels of total HELQ protein. In some cell lines, this antibody cross-reacts with a 200 kDa band of unknown identity by western blot.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro1091 of human HELQ protein.
DNA interstrand crosslinks (ICLs) are toxic DNA lesions caused by environmental agents as well as some chemotherapeutic drugs. These lesions are repaired via multiple DNA repair pathways. HELQ (also known as HEL308) is a 3’-5’ DNA helicase that colocalizes with stalled DNA replication forks in response to DNA damage, and contributes to the repair of ICL lesions through ATR signaling (1,2). HELQ interacts with various DNA repair proteins, including FANCD2, RAD51, RAD51 paralogs, and ATR (1-3). HELQ-deficient mice exhibit reduced fertility as well as interstrand crosslink (ICL) repair defects, and are prone to tumors (3).
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