Anti-MIRO2 antibody [RM2077]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MIRO2 antibody [RM2077] (AB322962)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RHOT2 KO HeLa (RHOT2 knockout human cervical adenocarcinoma epithelial cell), ab265801 cells labelling MIRO2 with ab322962 at 1/500 (0.906 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 mitochondrial staining in wildtype HeLa cells (shown in green), showing no staining in RHOT2 knockout HeLa cells. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

anti-COX IV mouse monoclonal antibody - Mitochondrial Marker was used to counterstain tubulin at 1/200 2.5 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

-ve control 1 : ab322962 at 1/500 dilution, followed byab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution. -ve control 2 : anti-COX IV mouse monoclonal antibody - Mitochondrial Marker at 1/200 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MIRO2 antibody [RM2077] (AB322962)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling MIRO2 with ab322962 at 1/100 (4.53 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon carcinoma. The section was incubated with ab322962 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-MIRO2 antibody [RM2077] (AB322962)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling MIRO2 with ab322962 at 1/100 (4.53 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon. The section was incubated with ab322962 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-MIRO2 antibody [RM2077] (AB322962)

Immunohistochemical analysis of paraffin-embedded Human pancreatic carcinoma tissue labeling MIRO2 with ab322962 at 1/100 (4.53 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human pancreatic carcinoma. The section was incubated with ab322962 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-MIRO2 antibody [RM2077] (AB322962)

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling MIRO2 with ab322962 at 1/100 (4.53 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human breast carcinoma. The section was incubated with ab322962 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-MIRO2 antibody [RM2077] (AB322962)

Immunohistochemical analysis of paraffin-embedded Human endometrium tissue labeling MIRO2 with ab322962 at 1/100 (4.53 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human endometrium. The section was incubated with ab322962 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

• WB

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Western blot - Anti-MIRO2 antibody [RM2077] (AB322962)

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

Low expression : mouse lung (PMID : 15218247).

The identity of the band lower than 75 kDa is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lane 1 : 10 seconds; Lanes 2-3 : 81 seconds; Lanes 4-6 : 37 seconds.

All lanes:

Western blot - Anti-MIRO2 antibody [RM2077] (ab322962) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 3:

MEF (mouse embryo fibroblast) whole cell lysate at 20 µg

Lane 4:

Mouse heart tissue lysate at 20 µg

Lane 5:

Mouse lung tissue lysate at 20 µg

Lane 6:

Mouse testis 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

Observed band size: 80 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-MIRO2 antibody [RM2077] (AB322962)

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

The samples were run on a Bis-Tris gel under reducing conditions.

Western blot : Anti-MIRO2 antibody (ab322962) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 loading control staining at 1/20000 dilution, shown in magenta.

In Western blot, ab322962 was shown to bind specifically to MIRO2. Target of interest was observed at 80 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in MIRO2 knockout cell line (lane 2, knockout cell line ab265801 / knockout cell lysate ab257639). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

All lanes:

Western blot - Anti-MIRO2 antibody [RM2077] (ab322962) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Western blot - Human RHOT2 (MIRO2) knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-rhot2-miro2-knockout-hela-cell-lysate-ab257639'>ab257639</a>) at 20 µg

Lane 3:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 4:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (800CW) and Goat Anti-Mouse IgG H&L (680RD) at 1/20000 dilution

Observed band size: 80 kDa

false

• WB

Supplier Data

Western blot - Anti-MIRO2 antibody [RM2077] (AB322962)

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

The identity of the band lower than 75 kDa is unknown.

Exposure time : Lane 1 : 81 seconds; Lane 2 : 180 seconds.

All lanes:

Western blot - Anti-MIRO2 antibody [RM2077] (ab322962) at 1/1000 dilution

Lane 1:

Human hippocampus tissue lysate at 20 µg

Lane 2:

Human testis tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 80 kDa

false

• WB

Supplier Data

Western blot - Anti-MIRO2 antibody [RM2077] (AB322962)

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

All lanes:

Western blot - Anti-MIRO2 antibody [RM2077] (ab322962) at 1/1000 dilution

Lane 1:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 20 µg

Lane 2:

Rat testis 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

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-MIRO2 antibody [RM2077] (AB322962)

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

This antibody does not cross-react with recombinant human MIRO1 by western blot.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-MIRO2 antibody [RM2077] (ab322962) at 1/1000 dilution

Lane 1:

His-tagged human MIRO2 fragment at 10 ng

Lane 2:

His-tagged human MIRO1 fragment at 10 ng

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

Observed band size: 21 kDa,23 kDa

false

Exposure time: 15s