Anti-HMGCS2 antibody [EPR26978-39]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-HMGCS2 antibody [EPR26978-39] (AB305227)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HCT116 (human colorectal carcinoma epithelial cell, Left) / Caco-2 (human colorectal adenocarcinoma epithelial cell, Right) cells labelling HMGCS2 with ab305227 at 1/500 dilution (0.1ug) (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. Negative control : HCT116 (PMID : 28468827).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGCS2 antibody [EPR26978-39] (AB305227)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling HMGCS2 with ab305227 at 1/2000 (0.237 µg/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Negative control : no staining on human cerebrum (PMID : 7893153, PMID : 22523078, PMID : 23943793, PMID : 24391449). The section was incubated with ab305227 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGCS2 antibody [EPR26978-39] (AB305227)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling HMGCS2 with ab305227 at 1/2000 (0.237 µg/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Cytoplasmic staining on human colon (PMID : 35422042, PMID : 31355161). The section was incubated with ab305227 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGCS2 antibody [EPR26978-39] (AB305227)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling HMGCS2 with ab305227 at 1/2000 (0.237 µg/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Cytoplasmic staining on human liver (PMID : 31779269). The section was incubated with ab305227 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. 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-HMGCS2 antibody [EPR26978-39] (AB305227)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Caco-2 (human colorectal adenocarcinoma epithelial cell) cells labelling HMGCS2 with ab305227 at 1/500 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing mitochondrial staining in Caco-2 cells. Negative control : HCT 116 (PMID : 28468827). The image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab33985 Anti-COX IV mouse monoclonal antibody - Mitochondrial Marker was used to counterstain tubulin at 1/1000 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 1/1000 dilution.

• IP

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Immunoprecipitation - Anti-HMGCS2 antibody [EPR26978-39] (AB305227)

HMGCS2 was immunoprecipitated from 0.35 mg Caco-2 (human colorectal adenocarcinoma epithelial cell), whole cell lysate 10 μg with ab305227 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab305227 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : Caco-2 (human colorectal adenocarcinoma epithelial cell), whole cell lysate 10 μg Lane 2 : ab305227 IP in Caco-2 whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab305227 in Caco-2 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 180 seconds

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Immunoprecipitation - Anti-HMGCS2 antibody [EPR26978-39] (ab305227) at 1/30 dilution

All lanes:

Caco-2 (human colorectal adenocarcinoma epithelial cell)

Secondary

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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

Observed band size: 50 kDa

false

Exposure time: 180s

• WB

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Western blot - Anti-HMGCS2 antibody [EPR26978-39] (AB305227)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Negative control : HCT 116 (PMID : 28468827) Exposure time : lanes 1-2 : 6 seconds lanes 3-5 : 180 seconds

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Western blot - Anti-HMGCS2 antibody [EPR26978-39] (ab305227) at 1/1000 dilution

Lane 1:

Human liver tissue lysate at 20 µg

Lane 2:

Human colon tissue lysate at 20 µg

Lane 3:

COLO 205 (human colon adenocarcinoma tumor ascitic epithelial cell), whole cell lysate at 20 µg

Lane 4:

HCT 116 (human colorectal carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 5:

Caco-2 (human colorectal adenocarcinoma epithelial cell), 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: 50 kDa

false

• Dot

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Dot Blot - Anti-HMGCS2 antibody [EPR26978-39] (AB305227)

Dot blot analysis of HMGCS2 using ab305227 at 1 : 1000 (0.473 μg/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution. Lane 1 : His-tagged human HMGCS1 recombinant protein 10 ng Lane 2 : MYC/DDK tagged human HMGCS2 recombinant protein 10 ng Exposure time : 180 seconds Blocking and diluting buffer and concentration : 5% NFDM/TBST