All lanes : Anti-Acetyl CoA synthetase antibody (ab66038) at 1 µg/mlLane 1 : Liver (Human) Tissue Lysate - adult normal tissue (ab29889)Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate Lane 3 : U-87 MG (Human glioblastoma astrocytoma) Whole Cell LysateLane 4 : Colon tissue lysate (Human) Tissue Lysate - adult normal tissue (ab30051)Lane 5 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate (ab3950)Lysates/proteins at 10 µg per lane.SecondaryGoat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilutionPerformed under reducing conditions.
![Acetyl CoA synthetase was immunoprecipitated using 0.5mg HepG2 whole cell extract, 5µg of Rabbit polyclonal to Acetyl CoA synthetase and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, HepG2 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab66038.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 79kDa: Acetyl CoA synthetase.](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_1/1653_Acetyl-CoA-synthetase-Primary-antibodies-ab66038-11.jpg)
Acetyl CoA synthetase was immunoprecipitated using 0.5mg HepG2 whole cell extract, 5µg of Rabbit polyclonal to Acetyl CoA synthetase and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, HepG2 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab66038.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 79kDa: Acetyl CoA synthetase.
IHC image of Acetyl CoA synthetase staining in human liver carcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab66038, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
ICC/IF image of ab66038 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab66038, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) HeLa cells at 1µg/ml.