Anti-ITM2B antibody [EPR27849-60] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ITM2B antibody [EPR27849-60] - BSA and Azide free (AB314655)

This data was developed using ab314654, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded human skeletal muscle tissue labeling ITM2B with ab314654 at 1/500 (1.024 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Low expression tissue : no staining on human skeletal muscle. The section was incubated with ab314654 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 was performed 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-ITM2B antibody [EPR27849-60] - BSA and Azide free (AB314655)

This data was developed using ab314654, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded human Alzheimer disease brain tissue labeling ITM2B with ab314654 at 1/500 (1.024 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on neurons and plaques in human Alzheimer disease brain. The section was incubated with ab314654 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 was performed 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-ITM2B antibody [EPR27849-60] - BSA and Azide free (AB314655)

This data was developed using ab314654, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling ITM2B with ab314654 at 1/500 (1.024 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human kidney. The section was incubated with ab314654 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 was performed 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-ITM2B antibody [EPR27849-60] - BSA and Azide free (AB314655)

This data was developed using ab314654, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling ITM2B with ab314654 at 1/500 (1.024 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human cerebrum. The section was incubated with ab314654 for30 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ITM2B antibody [EPR27849-60] - BSA and Azide free (AB314655)

This data was developed using ab314654, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SH-SY5Y (human neuroblastoma epithelial cell) cells labelling ITM2B with ab314654 at 1/100 (5.12 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1012 (2ug/ml) dilution (Green). Confocal image showing cytoplasmic staining in SH-SY5Y cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1012 (2ug/ml) dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-ITM2B antibody [EPR27849-60] - BSA and Azide free (AB314655)

This data was developed using ab314654, the same antibody clone in a different buffer formulation. ITM2B was immunoprecipitated from 0.35 mg SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate with ab314654 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314654 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate Lane 2 : ab314654 IP in SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314654 in SH-SY5Y whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-ITM2B antibody [EPR27849-60] (<a href='/en-us/products/primary-antibodies/itm2b-antibody-epr27849-60-ab314654'>ab314654</a>) at 1/30 dilution

All lanes:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 76s

• WB

Supplier Data

Western blot - Anti-ITM2B antibody [EPR27849-60] - BSA and Azide free (AB314655)

This data was developed using ab314654, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST Low expression : liver (PMID : 10391242) The expression molecular weight observed is consistent with what has been described in the literature (PMID : 24524963, PMID : 28131015) Band at 84 KDa is likely representing oligomers of ITM2B. 55 kDa pro-ITM2B and 42 kDa ITM2B band were ovserved. Bands beneath 42 kDa was cleavage fragment(PMID : 24524963). In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-ITM2B antibody [EPR27849-60] (<a href='/en-us/products/primary-antibodies/itm2b-antibody-epr27849-60-ab314654'>ab314654</a>) at 1/1000 dilution

Lane 1:

Human hippocampus tissue lysate at 20 µg

Lane 2:

Human cerebellum tissue lysate at 20 µg

Lane 3:

Human liver 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 1/100000 dilution

Observed band size: 84 kDa,55 kDa,17-42 kDa

false

Exposure time: 136s

• WB

Supplier Data

Western blot - Anti-ITM2B antibody [EPR27849-60] - BSA and Azide free (AB314655)

This data was developed using ab314654, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST Low expression : spleen, skeletal muscle(PMID : 12566161, PMID : 10391242) The expression molecular weight observed is consistent with what has been described in the literature (PMID : 24524963, PMID : 28131015) Band at 84 KDa is likely representing oligomers of ITM2B. 55 kDa pro-ITM2B and 42 kDa ITM2B band were ovserved. Bands beneath 42 kDa was cleavage fragment(PMID : 24524963). In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution. Exposure time : Lane 1-3 : 180 seconds; Lane 4, 5 : 125 seconds

All lanes:

Western blot - Anti-ITM2B antibody [EPR27849-60] (<a href='/en-us/products/primary-antibodies/itm2b-antibody-epr27849-60-ab314654'>ab314654</a>) at 1/1000 dilution

Lane 1:

Human hippocampus tissue lysate at 20 µg

Lane 2:

Human spleen tissue lysate at 20 µg

Lane 3:

Human skeletal muscle tissue lysate at 20 µg

Lane 4:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

HeLa (human epithelial cell line from cervical adenocarcinoma) whole cell 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 1/100000 dilution

Observed band size: 84 kDa,55 kDa,17-42 kDa

false