Anti-c-Kit antibody [RM1190]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Kit antibody [RM1190] (AB317843)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling c-Kit with ab317843 at 1/2000 (0.248 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mast cells in human colon. The section was incubated with ab317843 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-c-Kit antibody [RM1190] (AB317843)

Immunohistochemical analysis of paraffin-embedded A Wild-type HAP1 cell pellet B Human KIT knockout HAP1(human chronic myelogenous leukemia near-haploid cell) cell pellet tissue labeling c-Kit with ab317843 at 1/1000 (0.496 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on A : Wild-type HAP1 cell pellet, no staining on B : human KIT knockout HAP1 cell pellet. The section was incubated with ab317843 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-c-Kit antibody [RM1190] (AB317843)

Immunohistochemical analysis of paraffin-embedded Human seminoma tissue labeling c-Kit with ab317843 at 1/2000 (0.248 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human seminoma. The section was incubated with ab317843 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

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-c-Kit antibody [RM1190] (AB317843)

Flow cytometric analysis of Mouse bone marrow cells labelling c-Kit with ab317843 at 1/500 dilution (0.1 ug)/Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Mouse bone marrow are co-stained with Lineage cocktail conjugated FITC (530/30BP).
Gated on viable cell.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-c-Kit antibody [RM1190] (AB317843)

Flow cytometric analysis of Mouse bone marrow cells labelling c-Kit with ab317843 at 1/500 dilution (0.1ug)/Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti-Rabbit IgG (Alexa Fluor® 647, ab150083) at 1/5000 dilution was used as the secondary antibody.

Mouse bone marrow are co-stained with CD34 conjugated BV421 (440/50BP).
Gated on viable cell.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Kit antibody [RM1190] (AB317843)

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling c-Kit with ab317843 at 1/2000 (0.248 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse testis. The section was incubated with ab317843 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-c-Kit antibody [RM1190] (AB317843)

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling c-Kit with ab317843 at 1/2000 (0.248 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on scattered mast cells in rat colon. The section was incubated with ab317843 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-c-Kit antibody [RM1190] (AB317843)

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling c-Kit with ab317843 at 1/2000 (0.248 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat testis. The section was incubated with ab317843 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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-c-Kit antibody [RM1190] (AB317843)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized P815 (mouse mastocytoma mast cell) cells labelling c-Kit with ab317843 at 1/500 (0.992 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 cytoplasmic staining in P815 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Negative control : NIH/3T3 (PMID : 29622475)
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 2.5ug/ml dilution (Magenta). 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 1/1000 2 ug/mL dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-c-Kit antibody [RM1190] (AB317843)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling c-Kit with ab317843 at 1/2000 (0.248 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on scattered mast cells in mouse colon. The section was incubated with ab317843 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

• IP

Supplier Data

Immunoprecipitation - Anti-c-Kit antibody [RM1190] (AB317843)

c-Kit was immunoprecipitated from 0.35 mg P815 (mouse mastocytoma mast Cell) whole cell lysate with ab317843 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317843 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : P815 (mouse mastocytoma mast Cell) whole cell lysate
Lane 2 : ab317843 IP in P815 (mouse mastocytoma mast Cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317843 in P815 whole cell lysate

All lanes:

Immunoprecipitation - Anti-c-Kit antibody [RM1190] (ab317843) at 1/30 dilution

All lanes:

P815 (mouse mastocytoma mast Cell) whole cell lysate

Secondary

All lanes:

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

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-c-Kit antibody [RM1190] (AB317843)

Low expression : HL-60 (PMID : 29127384) and Jurkat (PMID : 22140461).

Negative control : NIH/3T3 (PMID : 29622475, PMID : 11522667)

The molecular weight observed is consistent with what has been described in the literature (PMID : 29176653, PMID : 23637779).

The identity of the lower MW bands at approximately 25-37kDa are unknown.

In Western blot, ab317843 was shown to bind specifically to c-Kit. Target of interest was observed at 120/140 kDa, in wild-type HAP1 cell lysates (lane 1) with no signal observed at this size in c-Kit knockout cell line (lane 2).

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

Exposure time : Lanes 1-2 : 180 seconds; Lanes 3-9 : 15 seconds

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

All lanes:

Western blot - Anti-c-Kit antibody [RM1190] (ab317843) at 1/1000 dilution

Lane 1:

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

Lane 2:

Kit HAP1 knockout whole cell lysate at 20 µg

Lane 3:

HEL (human erythroleukemia erythroblast) whole cell lysate at 20 µg

Lane 4:

Saos-2 (human osteosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

HL-60 (human acute promyelocytic leukemia promyeloblast) whole cell lysate at 20 µg

Lane 6:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 7:

P815 (mouse mastocytoma mast cell) whole cell lysate at 20 µg

Lane 8:

ES-D3 D3  (mouse blastocyst-derived embryonic stem cell) whole cell lysate at 20 µg

Lane 9:

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

Secondary

All lanes:

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

Observed band size: 120 kDa,140 kDa,36 kDa

false