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59355
Cathepsin B (D1C7Y) XP® Rabbit mAb (PE Conjugate)
Antibody Conjugates

Monoclonal Antibody - Cathepsin B (D1C7Y) XP® Rabbit mAb (PE Conjugate), UniProt ID P07858, Entrez ID 1508 #59355

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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).

To Purchase # 59355S

Supporting Data

REACTIVITY H M R
SENSITIVITY Endogenous
MW (kDa)
Isotype Rabbit IgG

Product Description

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.

Product Usage Information

Application Dilutions
Flow Cytometry 1:50

Storage:

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.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Specificity / Sensitivity

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

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the heavy chain subunit of human cathepsin B protein.

Background

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).

  1. Chan, S.J. et al. (1986) Proc Natl Acad Sci USA 83, 7721-5.
  2. Fong, D. et al. (1986) Proc Natl Acad Sci USA 83, 2909-13.
  3. Bever, C.T. et al. (1994) Neurology 44, 745-8.
  4. Hashimoto, Y. et al. (2001) Biochem Biophys Res Commun 283, 334-9.
  5. Halangk, W. et al. (2000) J Clin Invest 106, 773-81.
  6. Berquin, I.M. and Sloane, B.F. (1996) Adv Exp Med Biol 389, 281-94.
  7. Yan, S. et al. (1998) Biol Chem 379, 113-23.
  8. Vasiljeva, O. et al. (2006) Cancer Res 66, 5242-50.
  9. Campo, E. et al. (1994) Am J Pathol 145, 301-9.
  10. Foekens, J.A. et al. (1998) J Clin Oncol 16, 1013-21.
  11. Werle, B. et al. (1999) Br J Cancer 81, 510-9.
  12. Lah, T.T. et al. (2000) Clin Cancer Res 6, 578-84.
  13. Werle, B. et al. (2000) Cancer 89, 2282-91.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

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