All lanes : Anti-Acetyl Coenzyme A carboxylase alpha antibody [EPR23235-47] (ab269272) at 1/5000 dilution

Lane 1 : 293T (human embryonic kidney epithelial cell), whole cell lysate, in the loading buffer containing double DTT
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate, in the loading buffer containing double DTT
Lane 3 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate, in the loading buffer containing double DTT
Lane 4 : C2C12 (mouse myoblasts myoblast), whole cell lysate, in the loading buffer containing double DTT

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 265 kDa
Observed band size: 265 kDa

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

Exposure time: Lane 1, 4: 48 seconds; Lane 2, 3: 26 seconds.

Acetyl Coenzyme A carboxylase alpha was immunoprecipitated from 0.35 mg 293T (human embryonic kidney epithelial cell), whole cell lysate 10 µg with ab269272 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab269272 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: 293T whole cell lysate 10 µg.

Lane 2: ab269272 IP in 293T whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab269272 in 293T whole cell lysate.

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

Exposure time: 8 seconds.

Acetyl Coenzyme A carboxylase alpha was immunoprecipitated from 0.35 mg C2C12 (mouse myoblasts myoblast), whole cell lysate 10 µg with ab269272 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab269272 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: C2C12 whole cell lysate 10 µg.

Lane 2: ab269272 IP in C2C12 whole cell lysate 10 µg.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab269272 in C2C12 whole cell lysate.

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

Exposure time: 8 seconds.

 

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T (Human embryonic kidney epithelial cell) cells labelling Acetyl Coenzyme A carboxylase alpha with ab269272 at 1/500 dilution (0.1µg) (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, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized 293T cells labelling Acetyl Coenzyme A carboxylase alpha with ab269272 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing cytoplasmic staining in 293T cell line. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 2.5 µg/ml dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 2 µg/ml dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized C2C12 cells labelling Acetyl Coenzyme A carboxylase alpha with ab269272 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 2 µg/ml dilution (Green). Confocal image showing cytoplasmic staining in C2C12 cell line. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 2.5 µg/ml dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 2 µg/ml dilution.

Immunohistochemical analysis of paraffin-embedded rat lung tissue labeling Acetyl Coenzyme A carboxylase alpha with ab269272 at 1/500 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat lung (PMID: 17521700). The section was incubated with ab269272 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 ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded mouse lung tissue labeling Acetyl Coenzyme A carboxylase alpha with ab269272 at 1/500 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse lung (PMID: 17521700). The section was incubated with ab269272 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 ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human endometrial carcinoma tissue labeling Acetyl Coenzyme A carboxylase alpha with ab269272 at 1/500 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human endometrial carcinoma. The section was incubated with ab269272 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 ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling Acetyl Coenzyme A carboxylase alpha with ab269272 at 1/500 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on human breast carcinoma (PMID: 21415164). The section was incubated with ab269272 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 ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.