Anti-Cathepsin B 抗体 [EPR21033]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cathepsin B antibody [EPR21033] (AB214428)

Intracellular flow cytometric analysis of4% paraformaldehyde-fixed, 90% methanol-permeabilized RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cell line labeling Cathepsin B with ab214428 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cathepsin B antibody [EPR21033] (AB214428)

Immunofluorescent analysis of 100% methanol-fixed RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cells labeling Cathepsin B with ab214428 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in RAW 264.7 cells.

The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cathepsin B antibody [EPR21033] (AB214428)

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling Cathepsin B with ab214428 at 1/1000 dilution, followed by secondary Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Punctate cytoplasmic staining on rat kidney (PMID : 20668705; PMID : 26831567) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cathepsin B antibody [EPR21033] (AB214428)

Immunofluorescent analysis of 100% methanol-fixed PC-12 (rat adrenal gland pheochromocytoma cell line) cells labeling Cathepsin B with ab214428 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on PC-12 cells.

The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cathepsin B antibody [EPR21033] (AB214428)

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Cathepsin B with ab214428 at 1/1000 dilution, followed by secondary Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Punctate cytoplasmic staining on mouse kidney (PMID : 20668705; PMID : 26831567) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

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Western blot - Anti-Cathepsin B antibody [EPR21033] (AB214428)

Exposure times : Lanes 1,2 : 23 seconds; Lane 3 : 50 seconds; Lane 4 : 15 seconds; Lane 5 : 2 minutes.

Blocking/Dilution buffer : 5% NFDM/TBST.

The expression profile and molecular mass is consistent with what has been described in the literature (PMID : 20536394; PMID : 10447678).

All lanes:

Western blot - Anti-Cathepsin B antibody [EPR21033] (ab214428) at 1/1000 dilution

Lane 1:

Mouse kidney tissue lysate at 10 µg

Lane 2:

RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 3:

Rat thymus tissue lysate at 10 µg

Lane 4:

Rat kidney tissue lysate at 10 µg

Lane 5:

PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 38 kDa

Observed band size: 25 kDa,31 kDa,43 kDa

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• WB

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Western blot - Anti-Cathepsin B antibody [EPR21033] (AB214428)

Cathepsin B western blot using anti-Cathepsin B antibody [EPR21033] ab214428. Publication image and figure legend from Wu, Y., Whittaker, H. T., et al., 2021, PLoS One, PubMed 34292972.

ab214428 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab214428 please see the product overview.

Activity of cathepsin B in cortical lysates of 3- and 6- month-old mice.Total cortical proteins were used to determine enzyme activity, as the gain in fluorescence during the linear portion of the reaction curve relative to the wildtype mean. (A) Cortical relative cathepsin B activity at 3-months of age, graphed as group means ± SEM (n = 18 per genotype; (22 females and 14 males in total), revealed no statistically significant effects of dupCstb or sex (univariate ANOVA). (B) Cortical relative cathepsin B activity at 6-months of age, graphed as group means ± SEM, (dupCstb n = 7, wildtype n = 9; 7 females and 9 males in total), revealed no statistically significant effects of dupCstb or sex (univariate ANOVA). Recombinant human cathepsin B (R&D Systems, Cat. No. 953-CY-010) was used as a positive control. (C) Representative western blot probed with an anti-cathepsin B antibody that recognises pro-cathepsin B and cathepsin B heavy chain and an anti-β-actin antibody. (D) Protein band densities of pro-cathepsin B and cathepsin B heavy chain were quantified using ImageJ, normalised to β-actin, and are shown as cathepsin B/pro-cathepsin B ratio in the dupCstb group relative to the ratio in wildtype, graphed as group mean ± SEM (dupCstb n = 7, wildtype n = 9; 7 females and 9 males in total).

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