Anti-ProCathepsin B antibody [EPR24353-25] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ProCathepsin B antibody [EPR24353-25] - BSA and Azide free (AB282287)

This data was developed using ab270998, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling ProCathepsin B with ab270998 at 1/1000 (0.456 μg/mL) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Punctate cytoplasmic staining in human kidney. The section was incubated with ab270998 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ProCathepsin B antibody [EPR24353-25] - BSA and Azide free (AB282287)

This data was developed using ab270998, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human gastric adenocarcinoma tissue labelling ProCathepsin B with ab270998 at 1/1000 (0.456 μg/mL) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on human gastric adenocarcinoma. The section was incubated with ab270998 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ProCathepsin B antibody [EPR24353-25] - BSA and Azide free (AB282287)

This data was developed using ab270998, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human cervical carcinoma tissue labelling ProCathepsin B with ab270998 at 1/1000 (0.456 μg/mL) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining in human cervical carcinoma. The section was incubated with ab270998 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-ProCathepsin B antibody [EPR24353-25] - BSA and Azide free (AB282287)

This data was developed using ab270998, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized J774A.1 (Mouse reticulum cell sarcoma monocyte macrophage) cells labelling ProCathepsin B with ab270998 at 1/500 dilution (0.1μg)/ (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• WB

Collaborator

Western blot - Anti-ProCathepsin B antibody [EPR24353-25] - BSA and Azide free (AB282287)

This data was developed using ab270998, the same antibody clone in a different buffer formulation.

ab270998 was shown to react with CTSB in wild-type HAP1 cells in Western blot with loss of signal observed in a CTSB knockout cell line. Wild-type HAP1 and CTSB knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab270998 overnight at 4 °C at a 1/100 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-ProCathepsin B antibody [EPR24353-25] (<a href='/en-us/products/primary-antibodies/procathepsin-b-antibody-epr24353-25-ab270998'>ab270998</a>) at 1/100 dilution

Lane 1:

Wild-type HAP1 cell lysate at 30 µg

Lane 2:

CTSB knockout cell lysates at 30 µg

false

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-ProCathepsin B antibody [EPR24353-25] - BSA and Azide free (AB282287)

This data was developed using ab270998, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Daudi (Human Burkitt's lymphoma lymphoblast, Left)/ SK-MEL-28 (Human malignant melanoma, Right) cells labelling ProCathepsin B with ab270998 at 1/500 dilution (0.1μg)/ (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Negative control : Daudi.

• WB

Lab

Western blot - Anti-ProCathepsin B antibody [EPR24353-25] - BSA and Azide free (AB282287)

This data was developed using ab270998, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST. Lanes 1-5 : Merged signal (red and green). Green - ab270998 observed at 43 kDa. Red - loading control ab7291 observed at 50 kDa.

ab270998 Anti-ProCathepsin B antibody [EPR24353-25] was shown to specifically react with ProCathepsin B in wild-type HAP1 cells. Loss of signal was observed when knockout cell line was used. Wild-type and Cathepsin B knockout samples were subjected to SDS-PAGE. ab270998 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated at room temperature for 2. 5 hours at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Negative control : Daudi.

All lanes:

Western blot - Anti-ProCathepsin B antibody [EPR24353-25] (<a href='/en-us/products/primary-antibodies/procathepsin-b-antibody-epr24353-25-ab270998'>ab270998</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

Cathepsin B knockout HeLa cell lysate at 20 µg

Lane 3:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

SK-MEL-28 (human malignant melanoma ) whole cell lysate at 20 µg

Lane 5:

Daudi (human burkitts lymphoma lymphoblast) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 38 kDa

Observed band size: 43 kDa

false

• WB

Lab

Western blot - Anti-ProCathepsin B antibody [EPR24353-25] - BSA and Azide free (AB282287)

This data was developed using ab270998, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Exposure time : 10 seconds.

All lanes:

Western blot - Anti-ProCathepsin B antibody [EPR24353-25] (<a href='/en-us/products/primary-antibodies/procathepsin-b-antibody-epr24353-25-ab270998'>ab270998</a>) at 1/1000 dilution

Lane 1:

Mouse heart tissue lysate at 20 µg

Lane 2:

Mouse kidney tissue lysate at 20 µg

Lane 3:

Rat heart tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 38 kDa

Observed band size: 43 kDa

false

• WB

Lab

Western blot - Anti-ProCathepsin B antibody [EPR24353-25] - BSA and Azide free (AB282287)

This data was developed using ab270998, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This antibody specifically recognises the non-mature/full length form - ProCathepsin B.

Exposure time : 3 minutes.

All lanes:

Western blot - Anti-ProCathepsin B antibody [EPR24353-25] (<a href='/en-us/products/primary-antibodies/procathepsin-b-antibody-epr24353-25-ab270998'>ab270998</a>) at 1/5000 dilution

Lane 1:

His-tagged human Cathepsin B recombinant protein aa80-339

Lane 2:

His-tagged human Cathepsin B recombinant protein aa18-339

Secondary

All lanes:

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

Predicted band size: 38 kDa

false

• WB

Lab

Western blot - Anti-ProCathepsin B antibody [EPR24353-25] - BSA and Azide free (AB282287)

This data was developed using ab270998, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : Daudi.

Exposure time : Lanes 1-6 : 158 seconds; Lane 7 : 10 seconds.

All lanes:

Western blot - Anti-ProCathepsin B antibody [EPR24353-25] (<a href='/en-us/products/primary-antibodies/procathepsin-b-antibody-epr24353-25-ab270998'>ab270998</a>) at 1/1000 dilution

Lane 1:

SK-MEL-28 (human malignant melanoma) whole cell lysate at 20 µg

Lane 2:

U266B1 (human multiple myeloma b lymphocyte) whole cell lysate at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Daudi (human burkitts lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 5:

Mouse thymus tissue lysate at 20 µg

Lane 6:

J774A.1 (mouse reticulum cell sarcoma monocyte macrophage) whole cell lysate at 20 µg

Lane 7:

Rat thymus tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 38 kDa

Observed band size: 43 kDa

false