Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-HLA A antibody [EP1395Y] (ab52922)

Reviews (13)Q&A (8)References (54)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA A antibody [EP1395Y] (ab52922)
  • Western blot - Anti-HLA A antibody [EP1395Y] (ab52922)
  • Flow Cytometry - Anti-HLA A antibody [EP1395Y] (ab52922)
  • Immunocytochemistry/ Immunofluorescence - Anti-HLA A antibody [EP1395Y] (ab52922)
  • Western blot - Anti-HLA A antibody [EP1395Y] (ab52922)
  • Immunoprecipitation - Anti-HLA A antibody [EP1395Y] (ab52922)
  • Flow Cytometry - Anti-HLA A antibody [EP1395Y] (ab52922)
  • Immunocytochemistry/ Immunofluorescence - Anti-HLA A antibody [EP1395Y] (ab52922)
  • Western blot - Anti-HLA A antibody [EP1395Y] (ab52922)
  • Western blot - Anti-HLA A antibody [EP1395Y] (ab52922)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA A antibody [EP1395Y] (ab52922)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA A antibody [EP1395Y] (ab52922)
  • Immunoprecipitation - Anti-HLA A antibody [EP1395Y] (ab52922)
  • Anti-HLA A antibody [EP1395Y] (ab52922)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP1395Y] to HLA A
  • Suitable for: WB, IP, Flow Cyt, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Human

Overview

  • Product name

    Anti-HLA A antibody [EP1395Y]
    See all HLA A primary antibodies

  • Description

    Rabbit monoclonal [EP1395Y] to HLA A

  • Host species

    Rabbit

  • Tested Applications & Species

    Application Species
    Flow Cyt
    Human
    ICC/IF
    Human
    IHC-P
    Human
    IP
    Human
    WB
    Human
    See all applications and species data

  • Immunogen

    Synthetic peptide within Human HLA A aa 50-150. The exact sequence is proprietary.
    Database link: P04439

  • Positive control

    • IHC-P: Human tonsil tissue. ICC/IF: MCF7 and Raji cells. WB: A431, Jurkat, THP-1, A549, HL-60 and Raji cell lysates. IP: THP-1 and A549 cell lysates. Flow Cyt: Raji cells.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Properties

  • Form

    Liquid

  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.

  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: PBS, 40% Glycerol, 0.05% BSA
  • Concentration information loading...

  • Purity

    Protein A purified

  • Clonality

    Monoclonal

  • Clone number

    EP1395Y

  • Isotype

    IgG

  • Research areas

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab52922 in the following tested applications.

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

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
Flow Cyt
Human
ICC/IF
Human
IHC-P
Human
IP
Human
WB
Human
Application Abreviews Notes
WB (4)
1/2000. Predicted molecular weight: 41 kDa.

For unpurified use at 1/10000 - 1/50000.
IP
1/20.

For unpurified use at 1/30.
Flow Cyt (1)
1/10 - 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IHC-P (4)
1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

For unpurified use at 1/250 - 1/500.

See IHC antigen retrieval protocols.
ICC/IF (1)
1/100.

For unpurified use at 1/250 - 1/500.
Notes
WB
1/2000. Predicted molecular weight: 41 kDa.

For unpurified use at 1/10000 - 1/50000.
IP
1/20.

For unpurified use at 1/30.
Flow Cyt
1/10 - 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IHC-P
1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

For unpurified use at 1/250 - 1/500.

See IHC antigen retrieval protocols.
ICC/IF
1/100.

For unpurified use at 1/250 - 1/500.

Target

  • Relevance

    HLA-A belongs to the HLA class I heavy chain paralogues. This class I molecule is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). The heavy chain is anchored in the membrane. Class I molecules play a central role in the immune system by presenting peptides derived from the endoplasmic reticulum lumen. They are expressed in nearly all cells. The heavy chain is approximately 45 kDa and its gene contains 8 exons. Exon 1 encodes the leader peptide, exons 2 and 3 encode the alpha1 and alpha2 domains, which both bind the peptide, exon 4 encodes the alpha3 domain, exon 5 encodes the transmembrane region, and exons 6 and 7 encode the cytoplasmic tail. Polymorphisms within exon 2 and exon 3 are responsible for the peptide binding specificity of each class one molecule. Typing for these polymorphisms is routinely done for bone marrow and kidney transplantation. Hundreds of HLA-A alleles have been described.

  • Alternative names

    • Antigen presenting molecule antibody
    • HLA class I histocompatibility antigen, A 1 alpha chain antibody
    • HLAA antibody
    • Leukocyte antigen class I A antibody
    • Major histocompatibility complex, class I, A antibody
    • MHC class I antigen HLA A heavy chain antibody
    see all

Images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA A antibody [EP1395Y] (ab52922)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA A antibody [EP1395Y] (ab52922)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling HLA A with purified ab52922 at 1/100. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a goat anti-rabbit IgG H&L (HRP) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.
  • Western blot - Anti-HLA A antibody [EP1395Y] (ab52922)
    Western blot - Anti-HLA A antibody [EP1395Y] (ab52922)
    All lanes : Anti-HLA A antibody [EP1395Y] (ab52922) at 1/10000 dilution

    Lane 1 : Wild-type A431 whole cell lysate
    Lane 2 : HLA A knockout A431 whole cell lysate
    Lane 3 : A549 whole cell lysate
    Lane 4 : Jurkat whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 41 kDa
    Observed band size: 40 kDa
    why is the actual band size different from the predicted?



    Lanes 1 - 4: Merged signal (red and green). Green - ab52922 observed at 40 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

    ab52922 was shown to react with HLA-A in A431 wild-type cells in Western blot. Loss of signal was observed when HLA-A knockout sample was used. A431 wild-type and HLA-A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween®) before incubation with ab52922 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 10000 dilution and a 1 in 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 in 20000 dilution for 1 hour at room temperature before imaging.

  • Flow Cytometry - Anti-HLA A antibody [EP1395Y] (ab52922)
    Flow Cytometry - Anti-HLA A antibody [EP1395Y] (ab52922)
    Overlay histogram showing Raji cells stained with ab52922 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52922, 1/100) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Raji cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

  • Immunocytochemistry/ Immunofluorescence - Anti-HLA A antibody [EP1395Y] (ab52922)
    Immunocytochemistry/ Immunofluorescence - Anti-HLA A antibody [EP1395Y] (ab52922)

    Immunocytochemistry/Immunofluorescence analysis of Raji (human Burkitt's lymphoma) cells labelling HLA A with purified ab52922 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

    Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).

  • Western blot - Anti-HLA A antibody [EP1395Y] (ab52922)
    Western blot - Anti-HLA A antibody [EP1395Y] (ab52922)
    All lanes : Anti-HLA A antibody [EP1395Y] (ab52922) at 1/5000 dilution (purified)

    Lane 1 : THP-1 cell lysate at 20 µg
    Lane 2 : A549 cell lysate at 1/20 dilution

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 41 kDa



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

  • Immunoprecipitation - Anti-HLA A antibody [EP1395Y] (ab52922)
    Immunoprecipitation - Anti-HLA A antibody [EP1395Y] (ab52922)

    ab52922 (purified) at 1/20 immunoprecipitating HLA A in THP-1 whole cell lysate. 10 ug of cell lysate was present in the input. For western blotting, a HRP-conjugated Veriblot for IP Detection Reagent (ab131366) (1/10,000) was used for detection. A rabbit monoclonal IgG (ab172730) was used intead of ab128913 as a negative control (Lane 3).

    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

  • Flow Cytometry - Anti-HLA A antibody [EP1395Y] (ab52922)
    Flow Cytometry - Anti-HLA A antibody [EP1395Y] (ab52922)

    Flow Cytometry analysis of Raji cells labelling HLA A with purified ab52922 at 1/40 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

  • Immunocytochemistry/ Immunofluorescence - Anti-HLA A antibody [EP1395Y] (ab52922)
    Immunocytochemistry/ Immunofluorescence - Anti-HLA A antibody [EP1395Y] (ab52922)

    ICC/IF image of unpurified ab52922 stained MCF7 cells. The cells were 4% formaldehyde 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 (ab52922, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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.

  • Western blot - Anti-HLA A antibody [EP1395Y] (ab52922)
    Western blot - Anti-HLA A antibody [EP1395Y] (ab52922)
    Anti-HLA A antibody [EP1395Y] (ab52922) at 1/2000 dilution (purified) + HL-60 cell lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

    Predicted band size: 41 kDa



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

  • Western blot - Anti-HLA A antibody [EP1395Y] (ab52922)
    Western blot - Anti-HLA A antibody [EP1395Y] (ab52922)
    Anti-HLA A antibody [EP1395Y] (ab52922) at 1/10000 dilution + Raji cell lysate at 10 µg

    Secondary
    Goat anti rabbit IgG HRP conjugated at 1/2000 dilution

    Predicted band size: 41 kDa
    Observed band size: 41 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA A antibody [EP1395Y] (ab52922)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA A antibody [EP1395Y] (ab52922)This image is courtesy of an anonymous Abreview

    ab52922 staining HLA A in Human tonsil tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 3% H2O2 for 10 minutes at 25°C; antigen retrieval was by heat mediation in a citrate buffer, pH 6.0 . Samples were incubated with primary antibody (1/3000) for 20 minutes at 25°C. An undiluted HRP-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.

    See Abreview

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA A antibody [EP1395Y] (ab52922)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HLA A antibody [EP1395Y] (ab52922)

    Ab52922 at 1/250 dilution staining human tonsil; paraffin embedded.

  • Immunoprecipitation - Anti-HLA A antibody [EP1395Y] (ab52922)
    Immunoprecipitation - Anti-HLA A antibody [EP1395Y] (ab52922)

    ab52922 (purified) at 1/20 immunoprecipitating HLA A in A549 whole cell lysate. 10 ug of cell lysate was present in the input. For western blotting, a HRP-conjugated Veriblot for IP Detection Reagent (ab131366) (1/10,000) was used for detection. A rabbit monoclonal IgG (ab172730) was used intead of ab128913 as a negative control (Lane 3).

    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

  • Anti-HLA A antibody [EP1395Y] (ab52922)
    Anti-HLA A antibody [EP1395Y] (ab52922)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (54)

Publishing research using ab52922? Please let us know so that we can cite the reference in this datasheet.

ab52922 has been referenced in 54 publications.

  • Marie KL  et al. Melanoblast transcriptome analysis reveals pathways promoting melanoma metastasis. Nat Commun 11:333 (2020). PubMed: 31949145
  • Varanda AB  et al. Zebrafish Xenografts Unveil Sensitivity to Olaparib beyond BRCA Status. Cancers (Basel) 12:N/A (2020). PubMed: 32630796
  • Catanzaro V  et al. Gadolinium-Labelled Cell Scaffolds to Follow-up Cell Transplantation by Magnetic Resonance Imaging. J Funct Biomater 10:N/A (2019). PubMed: 31269673
  • Shukrun R  et al. NCAM1/FGF module serves as a putative pleuropulmonary blastoma therapeutic target. Oncogenesis 8:48 (2019). PubMed: 31477684
  • Visochek L  et al. The phenanthrene derivative PJ34 exclusively eradicates human pancreatic cancer cells in xenografts. Oncotarget 10:6269-6282 (2019). PubMed: 31692907
View all Publications for this product

Customer reviews and Q&As

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Flow Cytometry abreview for Anti-MHC class I antibody [EP1395Y]

 Below Average
Abreviews
abreview image
Application
Flow Cytometry
Sample
Human Cell (Fibrosarcoma HT1080)
Specification
Fibrosarcoma HT1080
Fixation
Paraformaldehyde
Permeabilization
No
Gating Strategy
no gating
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Submitted Nov 20 2009

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