Anti-MIRO2 抗体 [EPR29118-85] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MIRO2 antibody [EPR29118-85] - BSA and Azide free (AB320740)

This data was developed using ab320739, the same antibody clone in a different buffer formulation. 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 ab320739 at 1/50 (9.12 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. 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 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta). The Nuclear counterstain was DAPI (Blue). -ve control 1 : ab320739 at 1/50 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) antibody at 1/1000 (2 ug/ml) dilution. -ve control 2 : anti-COX IV mouse monoclonal antibody at 1/50 dilution, followed by 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-MIRO2 antibody [EPR29118-85] - BSA and Azide free (AB320740)

This data was developed using ab320739, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling MIRO2 with ab320739 at 1/100 (4.56 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Hematoxylin was used as a counterstain.

Positive staining on human human colon carcinoma. The section was incubated with ab320739 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MIRO2 antibody [EPR29118-85] - BSA and Azide free (AB320740)

This data was developed using ab320739, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human endometrium tissue labeling MIRO2 with ab320739 at 1/100 (4.56 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Hematoxylin was used as a counterstain.

Positive staining on human endometrium. The section was incubated with ab320739 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MIRO2 antibody [EPR29118-85] - BSA and Azide free (AB320740)

This data was developed using ab320739, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human endometrial carcinoma tissue labeling MIRO2 with ab320739 at 1/100 (4.56 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Hematoxylin was used as a counterstain.

Positive staining on human endometrial carcinoma. The section was incubated with ab320739 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument

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

Supplier Data

Western blot - Anti-MIRO2 antibody [EPR29118-85] - BSA and Azide free (AB320740)

This data was developed using ab320739, the same antibody clone in a different buffer formulation. Blocking buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of TBS. Diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBST The samples were run on a Bis-Tris gel under reducing conditions. Western blot : Anti-MIRO2 antibody (ab320739) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab320739 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 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. Exposure time : N/A

All lanes:

Western blot - Anti-MIRO2 antibody [EPR29118-85] (<a href='/products/primary-antibodies/miro2-antibody-epr29118-85-ab320739'>ab320739</a>) 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='/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 [EPR29118-85] - BSA and Azide free (AB320740)

This data was developed using ab320739, the same antibody clone in a different buffer formulation.

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

Lane 3 are applied with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 and lanes 1-2 are applied with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000.

Lanes 1-2 of this blot were developed using a high sensitivity ECL substrate.

The high-sensitivity ECL substrate allows for the detection of proteins in the mid-femtogram range.

Exposure time : Lanes 1-2 : 180 seconds; Lane 3 : 6 seconds.

All lanes:

Western blot - Anti-MIRO2 antibody [EPR29118-85] (<a href='/products/primary-antibodies/miro2-antibody-epr29118-85-ab320739'>ab320739</a>) at 1/1000 dilution

Lane 1:

Human testis tissue lysate at 20 µg

Lane 2:

Human hippocampus tissue lysate at 20 µg

Lane 3:

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

Secondary

Lanes 1 - 2:

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

Lane 3:

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

Observed band size: 80 kDa

false

• WB

Supplier Data

Western blot - Anti-MIRO2 antibody [EPR29118-85] - BSA and Azide free (AB320740)

This data was developed using ab320739, the same antibody clone in a different buffer formulation. 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 [EPR29118-85] (<a href='/products/primary-antibodies/miro2-antibody-epr29118-85-ab320739'>ab320739</a>) 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='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 20 kDa,21 kDa

false

Exposure time: 10s