Flow cytometric analysis of Daudi cells (blue) and SK-MEL-28 cells (green) using Cathepsin B (D1C7Y) XP® Rabbit mAb (PE Conjugate) (solid lines) or a concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed lines).
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This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Cathepsin B (D1C7Y) XP® Rabbit mAb #31718.
Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted July 2009
revised August 2019
Protocol Id: 407
Cathepsin B (D1C7Y) XP® Rabbit mAb (PE Conjugate) recognizes endogenous levels of total cathepsin B protein. This antibody detects the heavy chain subunit of cathepsin B.Species Reactivity:
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the heavy chain subunit of human cathepsin B protein.
Cathepsin B (CSTB), part of the papain family of proteases, is a widely expressed lysosomal cysteine endopeptidase (1,2). Cathepsin B is produced from a larger precursor form, pro-cathepsin B, which runs at approximately 44 kDa on SDS-PAGE, and is proteolytically processed and glycosylated to form a mature two-chain protein containing a heavy chain (running at 27 and 24 kDa) and a light chain (5 kDa). High levels of cathepsin B are found in macrophages and osteoclasts, as well as various types of cancer cells, including lung, colon, prostate, breast, and stomach. In addition, expression of cathepsin B has been associated with multiple sclerosis (3), rheumatoid arthritis (4), and pancreatitis (5). While generally localized to lysosomes, in cancer alterations can lead to its secretion (6). Its role in tumor progression is thought to involve promotion of basement membrane degradation, invasion and metastasis (7,8). Expression can correlate with poor prognosis for a variety of forms of cancer (9-13).
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