Key Product Details

Species Reactivity

Validated:

Human

Cited:

Human

Applications

Validated:

Immunohistochemistry, Western Blot, Simple Western, Immunoprecipitation

Cited:

Immunohistochemistry, Western Blot, Neutralization, Immunocytochemistry

Label

Unconjugated

Antibody Source

Polyclonal Goat IgG
View all Cystatin E/M Primary Antibodies »

Product Specifications

Immunogen

Mouse myeloma cell line NS0-derived recombinant human Cystatin E/M
Arg29-Met149
Accession # Q15828

Specificity

Detects human Cystatin E/M in direct ELISAs and Western blots. In direct ELISAs, approximately 25% cross-reactivity with recombinant mouse Cystatin E/M is observed.

Clonality

Polyclonal

Host

Goat

Isotype

IgG

Scientific Data Images for Human Cystatin E/M Antibody

Detection of Human Cystatin E/M antibody by Western Blot.

Detection of Human Cystatin E/M by Western Blot.

Western blot shows lysates of human skin tissue. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human Cystatin E/M Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1286) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for Cystatin E/M at approximately 13 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human Cystatin E/M antibody by Simple WesternTM.

Detection of Human Cystatin E/M by Simple WesternTM.

Simple Western lane view shows lysates of human skin tissue, loaded at 0.2 mg/mL. A specific band was detected for Cystatin E/M at approximately 13 kDa (as indicated) using 50 µg/mL of Goat Anti-Human Cystatin E/M Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1286) followed by 1:50 dilution of HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system. Non-specific interaction with the 230 kDa Simple Western standard may be seen with this antibody.

Detection of Human Cystatin E/M/CST6 by Western Blot

Detection of Human Cystatin E/M/CST6 by Western Blot

Cystatin E/M and C secretions from various cell lines: Common laboratory non-melanoma and established laboratory melanoma cell lines (top), and primary and metastatic melanoma cell lines established from patients, as well as skin control (bottom). (A) Equal amounts of serum free media were collected from 5 × 105 cells 48 h after changing from ordinary growth media and secreted proteins were concentrated by TCA-precipitation and subjected to SDS-PAGE and immunoblotting. The filters were stained with a cystatin E/M-specific (upper panels) or a cystatin C-specific (lower panel) antibody, respectively. (B) Inhibitory activity against legumain was measured in the conditioned media as residual legumain activity. A partially purified legumain fraction from rat kidney was mixed with conditioned media and the ability to cleave the substrate Z-Ala-Ala-Asn-NHMec was measured by fluorometry. Control bar (100%) represents activity in the rat legumain fraction without addition of conditioned media. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/20074384), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Cystatin E/M/CST6 by Western Blot

Detection of Cystatin E/M/CST6 by Western Blot

Cystatin E/M over-expression in melanoma cells suppressed legumain activity: A375 and MCC11 cells were transfected with pCST6 (closed bars) or empty pTracer vector (control; open bars), media were changed the following day, and serum-free conditioned media and cell lysates were collected 24 h later. (A) Immunoblots of cystatin E/M in conditioned media (CM) and lysates. Secreted proteins were TCA-precipitated prior to SDS-PAGE and immunoblotting. The filters were stained with a cystatin E/M-specific (upper and middle panels) or a beta -actin specific (lower panel; loading control) antibody, respectively. (B) Inhibitory activity against legumain was measured in the conditioned media as residual legumain activity using fluorometry, a fixed amount of partially purified legumain from rat kidney and the substrate Z-Ala-Ala-Asn-NHMec (A375, p < 0.0001; MCC11, p = 0.002). (C) Activity of legumain in cell lysates measured by cleavage of Z-Ala-Ala-Asn-NHMec (A375, p < 0.0001; MCC11, p = 0.0012). Error bars represent the standard error of the mean (SEM). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/20074384), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Human Cystatin E/M Antibody

Application
Recommended Usage

Immunohistochemistry

5-15 µg/mL
Sample: Immersion fixed paraffin-embedded sections of human skin

Immunoprecipitation

25 µg/mL
Sample: Conditioned cell culture medium spiked with Recombinant Human Cystatin E/M (Catalog # 1286-PI), see our available Western blot detection antibodies

Simple Western

50 µg/mL
Sample: Human skin tissue

Western Blot

1 µg/mL
Sample: Human skin tissue

Reviewed Applications

Read 1 review rated 5 using AF1286 in the following applications:

Formulation, Preparation, and Storage

Purification

Antigen Affinity-purified

Reconstitution

Reconstitute at 0.2 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Cystatin E/M

Cystatin E/M encoded by the CST6 gene is a member of family 2 of the cystatin superfamily (1, 2). It inhibits papain and cathepsin B, two of the cysteine proteases. Its mRNA was found in many tissues by the two groups who did initial cloning (1, 2). However, its protein was found only in skin and sweat glands by a third group (3). In addition to being a cysteine protease inhibitor, cystatin E/M is also a substrate for transglutaminases (3). It is required for viability and for correct formation of cornified layers in the epidermis and hair follicles, as ichq mice, with a null mutation in the cystatin E/M gene, have defects in epidermal cornification and die between 5 and 12 days of age (4). Cystatin E/M expression and function may not be limited to cutaneous epithelia. For example, it is found in rat brain and is induced during neuronal cell differentiation (5).

References

  1. Sotiropoulou, G. et al. (1997) J. Biol. Chem. 272:903.
  2. Ni, J. et al. (1997) J. Biol. Chem. 272:10853.
  3. Zeeuwen, P.L. et al. (2001) J. Invest. Dermatol. 116:693.
  4. Zeeuwen, P.L. et al. (2002) Hum. Mol. Genet. 11:2867.
  5. Hong, J. et al. (2002) J. Neurochem. 81:922.

Alternate Names

CST6

Entrez Gene IDs

1474 (Human); 73720 (Mouse)

Gene Symbol

CST6

UniProt

Q15828

Additional Cystatin E/M Products

Product Documents for Human Cystatin E/M Antibody

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Human Cystatin E/M Antibody

For research use only

Related Research Areas

Cysteine Proteases and Regulators

Citations for Human Cystatin E/M Antibody

Customer Reviews for Human Cystatin E/M Antibody (1)

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  • Name: Anonymous
    Application: Western Blot
    Sample Tested: See PMID 22902879
    Species: Human
    Verified Customer | Posted 01/05/2015

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