Western blot analysis of extracts from various tissues and cell lines using CD13/APN (E1Y7U) Rabbit mAb.
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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
CD13/APN (E1Y7U) Rabbit mAb recognizes endogenous levels of total CD13/APN protein.Species Reactivity:
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg90 of human CD13/APN protein.
Aminopeptidase N (APN, CD13) is a widely expressed, membrane-bound proteolytic enzyme that breaks down peptides during digestion, cleaves cell surface antigens during antigen presentation, and acts as a receptor for human viruses, including several coronaviruses. This multifunctional protein is implicated in the regulation of many biological processes, including angiogenesis, cell proliferation, cell migration, inflammation and immune response (1,2). APN was originally identified as the cell surface antigen CD13, which is expressed in myeloid lineage hematopoietic cells and myeloid leukemia (3). Identified substrates of aminopeptidase N include the angiotensin I-III peptide hormones, the opioid peptide met-enkephalin, and cytokines MCP-1 and MIP-1 (4). Abnormal APN protein expression is seen in various forms of cancer, with high APN expression associated with poor survival in colon cancer and non-small cell lung cancer and silenced APN expression related to poor prognosis in prostate cancer (5-7).
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