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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) #59355

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Flow Cytometry Image 1 - Cathepsin B (D1C7Y) XP® Rabbit mAb (PE Conjugate)

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
製品番号 サイズ 価格 在庫
59355S
100 µl  (50 tests)

Supporting Data

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

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

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 Dilution
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 Directly Conjugated Antibodies

A. Solutions and Reagents

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.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

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 for a full listing of cellular dyes validated for use in flow cytometry.

B. Fixation

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.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 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. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  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 primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised June 2020

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).

This product detects a SARS-CoV-2-related target for research into the mechanisms of the Novel Coronavirus, which has caused the COVID-19 pandemic.

  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.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

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