Overview

  • Product name

  • Description

    Rabbit polyclonal to ACSS2
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, ICC/IF, IHC-P, IPmore details
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Mouse, Rat
  • Immunogen

    Synthetic peptide corresponding to Human ACSS2 aa 650 to the C-terminus (C terminal) conjugated to keyhole limpet haemocyanin.
    (Peptide available as ab71894, ab71895)

  • Positive control

    • This antibody gave a positive signal in Human Liver and Human Colon Tissue lysates and in the following whole cell lysates: HepG2; U-87 MG; Caco 2.

Properties

Applications

Our Abpromise guarantee covers the use of ab66038 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use a concentration of 1 µg/ml. Detects a band of approximately 79 kDa (predicted molecular weight: 79 kDa).
ICC/IF Use a concentration of 1 µg/ml.
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IP Use at an assay dependent concentration.

Target

  • Function

    Activates acetate so that it can be used for lipid synthesis or for energy generation.
  • Sequence similarities

    Belongs to the ATP-dependent AMP-binding enzyme family.
  • Cellular localization

    Cytoplasm.
  • Information by UniProt
  • Database links

  • Alternative names

    • ACAS2 antibody
    • AceCS antibody
    • Acetate CoA ligase antibody
    • Acetate thiokinase antibody
    • Acetate--CoA ligase antibody
    • Acetyl CoA synthetase antibody
    • Acetyl Coenzyme A synthetase 2 (ADP forming) antibody
    • Acetyl coenzyme A synthetase cytoplasmic antibody
    • Acetyl-CoA synthetase antibody
    • Acetyl-coenzyme A synthetase antibody
    • ACS antibody
    • ACSA antibody
    • ACSA_HUMAN antibody
    • ACSS2 antibody
    • Acyl activating enzyme antibody
    • Acyl CoA synthetase short chain family member 2 antibody
    • Acyl-activating enzyme antibody
    • Acyl-CoA synthetase short-chain family member 2 antibody
    • Cytoplasmic acetyl coenzyme A synthetase antibody
    • cytoplasmic antibody
    • MYH7B antibody
    see all

Images

  • All lanes : Anti-ACSS2 antibody (ab66038) at 1 µg/ml

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : ACSS2 knockout HAP1 whole cell lysate
    Lane 3 : HepG2 whole cell lysate
    Lane 4 : U-87 MG whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 79 kDa
    Observed band size: 80 kDa
    why is the actual band size different from the predicted?



    Lanes 1 - 4: Merged signal (red and green). Green - ab66038 observed at 80 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab66038 was shown to recognize ACSS2 in wild-type HAP1 cells as signal was lost at the expected MW in ACSS2 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and ACSS2 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab66038 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • All lanes : Anti-ACSS2 antibody (ab66038) at 1 µg/ml

    Lane 1 : Human liver tissue lysate - total protein (ab29889)
    Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
    Lane 3 : U-87 MG (Human glioblastoma astrocytoma) Whole Cell Lysate
    Lane 4 : Human colon tissue lysate - total protein (ab30051)
    Lane 5 : Caco 2 whole cell lysate (ab3950)

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size: 79 kDa
    Observed band size: 79 kDa
    Additional bands at: 100 kDa, 38 kDa, 45 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 30 seconds
  • ACSS2 was immunoprecipitated using 0.5mg HepG2 whole cell extract, 5µg of Rabbit polyclonal to ACSS2 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: ACSS2.

  • IHC image of ACSS2 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.

References

This product has been referenced in:

  • Sun L  et al. Decreased expression of acetyl-CoA synthase 2 promotes metastasis and predicts poor prognosis in hepatocellular carcinoma. Cancer Sci 108:1338-1346 (2017). WB, IHC . Read more (PubMed: 28387999) »
  • Chen R  et al. Coordinate regulation of stress signaling and epigenetic events by Acss2 and HIF-2 in cancer cells. PLoS One 12:e0190241 (2017). Read more (PubMed: 29281714) »
See all 4 Publications for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (human fibroblast)
Total protein in input
500 µg
Immuno-precipitation step
Other - Trueblot from rockland
Specification
human fibroblast

Abcam user community

Verified customer

Submitted Aug 20 2013

Application
Western blot
Sample
Human Cell lysate - whole cell (human fibroblast)
Gel Running Conditions
Reduced Denaturing
Loading amount
40 µg
Specification
human fibroblast
Blocking step
Milk as blocking agent for 4 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jun 10 2013

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