Anti-ISG15 antibody [EPR24482-49]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ISG15 antibody [EPR24482-49] (AB285367)

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling ISG15 with ab285367 at 1/500 (0.998 ug/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) at Ready to use dilution.

Positive staining on human breast cancer (PMID : 18627608)

The section was incubated with ab285367 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).

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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ISG15 antibody [EPR24482-49] (AB285367)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type HAP1 cells treated with 1000 U/ml human IFN alpha 1 for 24 hours (B) Untreated wild-type HAP1 cells (C) ISG 15 knockout HAP1 cells treated with 1000U/ml human IFN alpha 1 for 24 hours (D) Untreated ISG 15 knockout HAP1 cells tissue labelling ISG15 with ab285367 at 1/500 (0.998 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on (A) HAP1 cells treated with IFN alpha 1, no staining on (B) untreated wild-type HAP1 cells. Positive staining on a few cells of (C) ISG15 knockout HAP1 cells treated with 1000 U/ml IFN alpha 1, no staining on (D) untreated ISG 15 knockout HAP1 cells. The section was incubated with ab285367 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)

Lab

Flow Cytometry (Intracellular) - Anti-ISG15 antibody [EPR24482-49] (AB285367)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) (Right)/ ISG15 knockout HAP1 cells (Left).
HAP1 treated with 1000U/ml hIFN alpha 1 for 24 hours (Red) / Untreated control (Green) cells labelling ISG15 with ab285367 at 1/50 dilution (1ug)(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, ab150081) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ISG15 antibody [EPR24482-49] (AB285367)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized parental HAP1+hIFN-a1(1000U/ml 24h)(-) parental HAP1+hIFN-a1(1000U/ml 24h)(+) ISG15 KO HAP1+hIFN-a1(1000U/ml 24h)(-) ISG15 KO HAP1+hIFN-a1(1000U/ml 24h)(+) cells labelling ISG15 with ab285367 at 1/100 (4.99 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing increased cytoplasmic staining in HAP1 cells treated with hIFN-a1(1000 U/ml) for 24 h, and no staining in ISG15 knockout HAP1 cells treated with hIFN-a1(1000 U/ml) for 24 h is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ISG15 antibody [EPR24482-49] (AB285367)

Immunohistochemical analysis of paraffin-embedded Human gastric cancer tissue labelling ISG15 with ab285367 at 1/500 (0.998 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Positive staining on human gastric cancer (PMID : 30842212). The section was incubated with ab285367 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

• IP

Lab

Immunoprecipitation - Anti-ISG15 antibody [EPR24482-49] (AB285367)

ISG15 was immunoprecipitated from 0.35 mg Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) treated with 1000U/ml human IFN alpha 1 for 24 hours whole cell lysate 10 μg with ab285367 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab285367 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) treated with 1000U/ml human IFN alpha 1 for 24 hours whole cell lysate 10 μg

Lane 2 : ab285367 IP in Wild-type HAP1 treated with 1000U/ml human IFN alpha 1 for 24 hours whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab285367 in Wild-type HAP1 treated with 1000U/ml human IFN alpha 1 for 24 hours whole cell lysate

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

Exposure time : 7.75 second

All lanes:

Immunoprecipitation - Anti-ISG15 antibody [EPR24482-49] (ab285367)

Predicted band size: 18 kDa

false

• WB

Lab

Western blot - Anti-ISG15 antibody [EPR24482-49] (AB285367)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lanes 1-6 : Merged signal (red and green). Green - ab285367 observed at 14 kDa. Red - loading control ab8245 observed at 36 kDa.

ab285367 Anti-ISG15 antibody [EPR24482-49] was shown to specifically react with ISG15 in IFN alpha 1 induced wild-type HAP1 cells. Loss of signal was observed when knockout cell line was used. Wild-type and ISG15 knockout samples were subjected to SDS-PAGE. ab285367 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-ISG15 antibody [EPR24482-49] (ab285367) at 1/1000 dilution

Lane 1:

Untreated wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 2:

Wild-type HAP1 treated with 1000U/ml human IFN alpha 1 for 24 hours whole cell lysate at 20 µg

Lane 3:

Untreated ISG 15 knockout HAP1 whole cell lysate at 20 µg

Lane 4:

ISG 15 knockout HAP1 treated with 1000U/ml human IFN alpha 1 for 24 hours whole cell lysate at 20 µg

Lane 5:

Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

HeLa treated with 10 ng/ml human IFN alpha 1 for 16 hours 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: 18 kDa

false