Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human TNFRSF8/CD30 protein (hCD30-Myc/DDK; +), using TNFRSF8/CD30 Antibody (upper), DYKDDDDK Tag Antibody #2368 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from various cell lines using TNFRSF8/CD30 Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, TNFRSF8/CD30 protein is not detected in either HPB-ALL or U-937 cells. KARPAS-299 cell line source: Dr. Abraham Karpas at the University of Cambridge.
Western blot analysis of extracts from KARPAS-299 cells, untreated (-) or treated with TPA #4174 (200nM, 90min; +), using TNFRSF8/CD30 Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). KARPAS-299 cell line source: Dr. Abraham Karpas at the University of Cambridge.
|MW (kDa)||90 (precursor), 120 (mature)|
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.
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
TNFRSF8/CD30 Antibody recognizes endogenous levels of total TNFRSF8/CD30 protein.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human TNFRSF8/CD30 protein. Antibodies are purified by protein A and peptide affinity chromatography.
TNFRSF8/CD30 is a type-I transmembrane glycoprotein that is a member of the TNFR superfamily. CD30 is synthesized as a precursor protein that undergoes extensive posttranslational modification before becoming embedded in the plasma membrane as a 120-kDa transmembrane protein (1,2). The expression of CD30 is upregulated in activated T-cells and may trigger costimulatory signaling pathways upon its engagement (3,4). While its expression is normally restricted to subsets of activated T-cells and B-cells, CD30 expression is robustly upregulated in hematologic malignancies, such as Hodgkin’s lymphoma (HL), anaplastic large cell lymphoma (ALCL), and adult T-cell leukemia, thus making it an attractive target for therapeutic intervention (5,6). Research studies have suggested that in certain disease contexts, CD30 recruits TRAF2 and TRAF5 adaptor proteins to drive NF-kappa B activation, aberrant cell growth, and cytokine production (7-9). CD30 signaling is also regulated by TACE-dependent proteolytic cleavage of its ectodomain, which results in reduced CD30L-dependent activation of CD30+ cells (10, 11).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.
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