Validated using a knockout cell line

Anti-ABCA1 antibody [HJ1] (ab66217)

Reviews (3)Q&A (2)References (6)

Key features and details

  • Mouse monoclonal [HJ1] to ABCA1
  • Suitable for: WB, Flow Cyt, IHC-P
  • Knockout validated
  • Reacts with: Mouse, Rat, Human
  • Isotype: IgG2b

Overview

  • Product name

    Anti-ABCA1 antibody [HJ1]
    See all ABCA1 primary antibodies

  • Description

    Mouse monoclonal [HJ1] to ABCA1

  • Host species

    Mouse

  • Tested applications

    Suitable for: WB, Flow Cyt, IHC-Pmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Recombinant fragment corresponding to ABCA1 (N terminal). 50 kDa N-terminal extracellular loop of ABCA1
    Database link: O95477

  • Positive control

    • IHC-P: Liver parenchyma and liver tissue. Flow: HepG2 cells. WB: Hap1 whole cell lysate.

  • General notes

    This monoclonal antibody to ABCA1 has been knockout validated in Western blot. The expected band for ABCA1 was observed in wild type cells and the band was not seen in knockout cells, although other non-specific bands were also seen. The data are shown on this datasheet.

    This antibody clone is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

Our Abpromise guarantee covers the use of ab66217 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 254 kDa (predicted molecular weight: 254 kDa).

Abcam recommends using 3% Milk as the blocking agent.
Flow Cyt Use 2µg for 106 cells.

(methanol fixed cells)

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
IHC-P Use a concentration of 0.2 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Target

  • Function

    cAMP-dependent and sulfonylurea-sensitive anion transporter. Key gatekeeper influencing intracellular cholesterol transport.

  • Tissue specificity

    Widely expressed, but most abundant in macrophages.

  • Involvement in disease

    Defects in ABCA1 are a cause of high density lipoprotein deficiency type 1 (HDLD1) [MIM:205400]; also known as analphalipoproteinemia or Tangier disease (TGD). HDLD1 is a recessive disorder characterized by absence of high density lipoprotein (HDL) cholesterol from plasma, accumulation of cholesteryl esters, premature coronary artery disease (CAD), hepatosplenomegaly, recurrent peripheral neuropathy and progressive muscle wasting and weakness.
    Defects in ABCA1 are a cause of high density lipoprotein deficiency type 2 (HDLD2) [MIM:604091]; also known as familial hypoalphalipoproteinemia (FHA). HDLD2 is inherited as autosomal dominant trait. It is characterized by moderately low HDL cholesterol, predilection toward premature coronary artery disease (CAD) and a reduction in cellular cholesterol efflux.

  • Sequence similarities

    Belongs to the ABC transporter superfamily. ABCA family.
    Contains 2 ABC transporter domains.

  • Domain

    Multifunctional polypeptide with two homologous halves, each containing an hydrophobic membrane-anchoring domain and an ATP binding cassette (ABC) domain.

  • Post-translational
    modifications

    Phosphorylation on Ser-2054 regulates phospholipid efflux.
    Palmitoylation by DHHC8 is essential for membrane localization.

  • Cellular localization

    Membrane.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • ABC 1 antibody
    • ABC Transporter 1 antibody
    • ABC-1 antibody
    • ABC1 antibody
    • ABCA 1 antibody
    • ABCA1 antibody
    • ABCA1_HUMAN antibody
    • ATP binding Cassette 1 antibody
    • ATP binding cassette sub family A ABC1 member 1 antibody
    • ATP binding cassette sub family A member 1 antibody
    • ATP binding Cassette Transporter 1 antibody
    • ATP-binding cassette 1 antibody
    • ATP-binding cassette sub-family A member 1 antibody
    • ATP-binding cassette transporter 1 antibody
    • CERP antibody
    • Cholesterol efflux regulatory protein antibody
    • FLJ14958 antibody
    • HDLDT1 antibody
    • Membrane bound antibody
    • MGC164864 antibody
    • MGC165011 antibody
    • TD antibody
    • TGD antibody
    see all

Images

  • Western blot - Anti-ABCA1 antibody [HJ1] (ab66217)
    Western blot - Anti-ABCA1 antibody [HJ1] (ab66217)
    All lanes : Anti-ABCA1 antibody [HJ1] (ab66217) at 1 µg/ml

    Lane 1 : Brain (Mouse) Tissue Lysate
    Lane 2 : Brain (Rat) Tissue Lysate
    Lane 3 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
    Lane 4 : Liver (Mouse) Tissue Lysate
    Lane 5 : Liver (Rat) Tissue Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size: 254 kDa
    Observed band size: 254 kDa
    Additional bands at: 70 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 4 minutes


    Abcam recommends using milk as the blocking agent. This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% Milk before being incubated with ab66217 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ABCA1 antibody [HJ1] (ab66217)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ABCA1 antibody [HJ1] (ab66217)
    ab66217 (4µg/ml) staining ABCA1 in human liver using an automated system (DAKO Autostainer Plus). Using this protocol there is moderate cell membrane staining throughout the liver parenchyma.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ABCA1 antibody [HJ1] (ab66217)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ABCA1 antibody [HJ1] (ab66217)
    IHC image of ABCA1 staining in human liver 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 ab66217, 0.2µg/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.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
  • Flow Cytometry - Anti-ABCA1 antibody [HJ1] (ab66217)
    Flow Cytometry - Anti-ABCA1 antibody [HJ1] (ab66217)
    Overlay histogram showing HepG2 cells stained with ab66217 (red line). The cells were fixed with methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab66217, 2µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in HepG2 cells fixed with 4% paraformaldehyde (10 min) used under the same conditions.

    Please note that Abcam does not have data for use of this antibody on non-fixed cells. We welcome any customer feedback.
  • Western blot - Anti-ABCA1 antibody [HJ1] (ab66217)
    Western blot - Anti-ABCA1 antibody [HJ1] (ab66217)

    Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: Empty
    Lane 3: ABCA1 knockout (KO) HAP1 whole cell lysate (20 µg)

     

    Lanes 1 - 3: Merged signal (red and green). Green - ab66217 observed at 240 kDa. Red - loading control, ab176560, observed at 50 kDa.

     

    ab66217 detected the expected band for ABCA1 in wild type HAP1 cells and the band was not seen in ABCA1 knockout HAP1 cells, although additional non-specific bands were also seen. Wild-type and ABCA1 knockout samples were subjected to SDS-PAGE. Ab66217 and ab176560 (Rabbit anti alpha Tubulin loading control) were incubated overnight at 4°C at 1 µg/ml and 1/10000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

References

Publishing research using ab66217? Please let us know so that we can cite the reference in this datasheet.

ab66217 has been referenced in 6 publications.

  • Liu J  et al. ZAP70 deficiency promotes reverse cholesterol transport through MAPK/ERK pathway in Jurkat cell. Mol Immunol 107:21-28 (2019). PubMed: 30639475
  • Yang S  et al. Polysaccharide IV fromLycium barbarum L. Improves Lipid Profiles of Gestational Diabetes Mellitus of Pregnancy by Upregulating ABCA1 and Downregulating Sterol Regulatory Element-Binding Transcription 1viamiR-33. Front Endocrinol (Lausanne) 9:49 (2018). PubMed: 29527188
  • Sandoval-Hernández AG  et al. Role of Liver X Receptor in AD Pathophysiology. PLoS One 10:e0145467 (2015). WB ; Mouse . PubMed: 26720273
  • Wu H  et al. Complete morphologies of basal forebrain cholinergic neurons in the mouse. Elife 3:e02444 (2014). IHC-FrFl . PubMed: 24894464
  • Martinez MN  et al. Obesity and altered glucose metabolism impact HDL composition in CETP transgenic mice: a role for ovarian hormones. J Lipid Res 53:379-89 (2012). WB ; Mouse . PubMed: 22215797
  • Fryirs MA  et al. Effects of high-density lipoproteins on pancreatic beta-cell insulin secretion. Arterioscler Thromb Vasc Biol 30:1642-8 (2010). WB ; Rat . PubMed: 20466975

Customer reviews and Q&As

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1-5 of 5 Abreviews or Q&A

Question

Thank you for your kind replacement of the antibody! I was able to test it on human prostate cancer cell line PC3 lysed with RIPA buffer (last lane of the samples indicated by arrow) and my other prostate cancer cell lines prepared before. The membrane was first detected with your new antibody and then reprobed with FASN. As you can see from the images, your new antibody can only show a low molecular weight background band. FASN antibody clearly detects the target protein with several non-specific bands. The pictures in two different pages represents different exposure time. Therefore, I am afraid that your new antibody does not work either. If you can provide a positive control, I am willing to repeat experiment again. If your positive control works, it means that your antibodies work. I am happy to keep them and improve my sample preparation.

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Answer


Yes, I can send you as a one time exception a positive control lysate (rat liver (ab29413), https://www.abcam.com/liver-rat-whole-cell-lysate-normal-tissue-ab29413.html). This has been tested with ab66217 and gave strong bands.

Also I would suggest to check the gel after transfer as some of the high MW proteins might have not completely transfered onto the membrane. You can check this by staining the gel with Coomassie Blue. if you see bands in the gel at 200 kDa and higher, you might want to adjust your transfer conditions so that all high MW protein are being transfered.

Also, avoid boiling of your samples before loading them onto the gel. Warming your sample to 50-60 degree Celsius is sufficient for denaturation in loading buffer.

Again, FASN is a cytoplasmic protein and such is much easier to work with than ABCA1 which is very hydrophobic, glycosylated and of high MW.

This article and link might be of help as well: http://www.sciencedirect.com/science/article/pii/S0003269708006829
http://www.scientistsolutions.com/t14685-membrane+protein+western+blot.html

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Immunocytochemistry abreview for Anti-ABCA1 antibody [HJ1]

 Average
Abreviews
abreview image
Application
Immunocytochemistry
Sample
Mouse Cultured Cells (RAW 264.7)
Specification
RAW 264.7
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton-X100 in 2% BSA for 15min
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 22°C
Read More

Dr. Mahesh Shivananjappa

Verified customer

Submitted May 19 2012

Question

AbID: 66217, Anti-ABCA1 antibody [HJ1] Rating: Below Average Image: Anti-ABCA1 antibody [HJ1] for Western blot (Human) Sample: Species: Human Type: Cell lysate - other Loading amount: 25 µg Specification: Mononuclear Cell Preparations Application: Application: Western blot Gel Running Conditions: Reduced Denaturing (4-15%) Blocking step: Milk as blocking agent for 1 hour at 22°C Blocking Concentration: 5% Other detail: Dilution: 1/1000 Incubation time: 16 hours at 4°C Diluent: 5% BSA in PBST Secondary Antibody: Name: Non-Abcam Antibody was used: Donkey anti-mouse HRP Conjugation: HRP Dilution: 1/10000 Detection: Detection method: ECL+ Exposure: 5 minutes and 0 seconds Bands: Specific: No kDa Non-specific: 48, 100, 120 kDa Additional Data:

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Answer

Thank you for submitting your recent review of ab66217. We are sorry to see that the results were unsatisfactory. I'd like to follow up with you to offer a couple of suggestions that may help improve these results. However we do fully guarantee this antibody to work in tested species and applications, so I would be happy to replace this antibody or issue a credit or refund if you would prefer. Some lower molecular weight bands are often seen with ABCA1 antibodies, and though the identity is not known, it is possible that these are cleavage fragments. Since the molecular weight of the full length ABCA1 is around 254 kDa, and this protein is heavily glycosylated, it might be difficult to transfer to the membrane. Did you do a Ponceau stain to verify the transfer? There are a few protocol alterations that we recommend when transfering such a large protein: 1) Large proteins will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will discourage this. Lowering methanol in the transfer buffer also promotes swelling of the gel, allowing large proteins to transfer more easily. We recommend 10% with nitrocellulose or none if using a PVDF membrane. 2) Choose wet transfer overnight at 4°C instead of semi-dry transfer. I look forward to hearing from you. If you would like a replacement, credit, or refund, please send me your order number and I will be happy to arrange this for you.

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Western blot abreview for Anti-ABCA1 antibody [HJ1]

 Below Average
Abreviews
abreview image
Application
Western blot
Sample
Human Cell lysate - other (Mononuclear Cell Preparations)
Loading amount
25 µg
Specification
Mononuclear Cell Preparations
Gel Running Conditions
Reduced Denaturing (4-15%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Read More

Dr. Mahesh Shivananjappa

Verified customer

Submitted Nov 23 2011

Flow Cytometry abreview for Anti-ABCA1 antibody [HJ1]

 Average
Abreviews
abreview image
Application
Flow Cytometry
Sample
Human Cell (Platelets)
Specification
Platelets
Preparation
Cell harvesting/tissue preparation method: PL were isolated spinning Platelet rich plasma on Histopaque
Sample buffer: PBS
Fixation
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton-X100 in 2% BSA for 15min
Gating Strategy
Platelets
Read More

Abcam user community

Verified customer

Submitted Dec 22 2010

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