Western blot analysis of extracts from ZR-75-1 cells, untreated (-) or treated with a Cub1 antibody (+), using Phospho-CDCP1 (Tyr806) Antibody (upper) and CDCP1 Antibody #4115 (lower).
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
Phospho-CDCP1 (Tyr806) Antibody recognizes endogenous levels of CDCP1 protein only when phosphorylated at Tyr806. This antibody does not cross-react with CDCP1 phosphorylated at Tyr707, Tyr734, or Tyr743, but may cross-react with other phospho-tyrosine containing proteins.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Tyr806 of human CDCP1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
CUB domain containing protein 1 (CDCP1, SIMA135) is a putative stem cell marker shown in research studies to be highly expressed in some human cancer cells and in both typical and atypical (cancerous) colons (1). Expression of CDCP1 may be epigenetically regulated, as methylation of promoter CpG sequences results in decreased CDCP1 expression (2). The corresponding CDCP1 gene encodes a glycoprotein that acts as a complex, multidomain transmembrane antigen. CDCP1 has three extracellular CUB domains that may be involved in cell adhesion or extracellular matrix interactions (1,3). Src-family kinases may phosphorylate CDCP1 at five tyrosine residues within its cytoplasmic domain to provide a potential binding site for SH2 domain-containing proteins (3). CDCP1 is a putative hematopoietic stem cell marker (4,5).
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