Western blot analysis of extracts from HeLa and IGROV-1 cells using DPYD (D35A8) Rabbit mAb.
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 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
DPYD (D35A8) Rabbit mAb detects endogenous levels of total DPYD protein. The antibody also detects a 50-60 kDa band of unknown origin by western blot.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Mouse, Rat, Monkey, Dog
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human DPYD protein.
Dihydropyrimidine dehydrogenase (DPD, DPYD) catalyzes the initial and rate-limiting step in uracil and thymidine catabolism as well as catabolism of the chemotherapeutic drug 5-fluorouracil (5-FU) and its derivatives. DPYD deficiency, which results from mutations in the DPYD gene, causes errors in pyrimidine metabolism and potentially life-threatening side effects in cancer patients treated with 5-FU (reviewed in 1). As a result, ongoing work examines whether or how DPYD gene variation and protein expression can be used to predict 5-FU toxicity (1,2). Several genes that impart resistance to 5-FU were recently identified in human hepatocellular carcinoma (HCC). AEG-1, which is highly expressed in HCC, increases the expression of DPYD. DPYD is expressed more highly in HCC than in normal liver, and this is thought to be one mechanism of 5-FU resistance (3,4).
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