Recombinant Anti-HLA A antibody [EP1395Y] - BSA and Azide free (ab216653)
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 - BSA and Azide free
- Suitable for: Flow Cyt (Intra), IHC-P, WB, ICC/IF, IP
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-HLA A antibody [EP1395Y] - BSA and Azide free
See all HLA A primary antibodies -
Description
Rabbit monoclonal [EP1395Y] to HLA A - BSA and Azide free -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt (Intra), IHC-P, WB, ICC/IF, IPmore details -
Species reactivity
Reacts with: Human
Predicted to work with: Rat -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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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 (intra): Raji cells.
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General notes
ab216653 is the carrier-free version of ab52922.
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.
This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.
Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.
This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.
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
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C. Do Not Freeze. -
Storage buffer
pH: 7.20
Constituent: PBS -
Carrier free
Yes -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EP1395Y -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Conjugation kits
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Isotype control
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KO cell lines
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KO cell lysates
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Positive Controls
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab216653 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 |
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Flow Cyt (Intra) |
Use at an assay dependent concentration.
ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
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IHC-P |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 41 kDa.
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ICC/IF |
Use at an assay dependent concentration.
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IP |
Use at an assay dependent concentration.
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Notes |
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Flow Cyt (Intra)
Use at an assay dependent concentration. ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
WB
Use at an assay dependent concentration. Predicted molecular weight: 41 kDa. |
ICC/IF
Use at an assay dependent concentration. |
IP
Use at an assay dependent concentration. |
Target
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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. -
Database links
- SwissProt: P04439 Human
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Alternative names
- Antigen presenting molecule antibody
- HLA class I histocompatibility antigen, A 1 alpha chain antibody
- HLAA antibody
see all
Images
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All lanes : Anti-HLA A antibody [EP1395Y] (ab52922) at 1/10000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : HLA-A knockout A549 cell lysate
Lane 3 : Wild-type A431 cell lysate
Lane 4 : HLA-A knockout A431 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 41 kDa
Observed band size: 41 kDaThis data was developed using the same antibody clone in a different buffer formulation (ab52922).
Anti-HLA-A antibody [EP1395Y] (ab52922) staining at 1/10000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab52922 was shown to bind specifically to HLA-A. A band was observed at 41 kDa in wild-type A549 cell lysates with no signal observed at this size in HLA-A knockout cell line. To generate this image, wild-type and HLA-A knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
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All lanes : Anti-HLA A antibody [EP1395Y] (ab52922) at 1/10000 dilution
Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : HLA A knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : A549 (Human lung carcinoma cell line) whole cell lysate
Lane 4 : Jurkat (Human T cell leukemia cell line from peripheral blood) 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?This data was developed using the same antibody clone in a different buffer formulation (ab52922).
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.
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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, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution. 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).
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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, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution. 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).
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Intracellular 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).
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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).
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).
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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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).
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Ab52922 at 1/250 dilution staining human tonsil; paraffin embedded.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).
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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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).
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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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).
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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 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab52922).
Protocols
Datasheets and documents
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Datasheet download
Certificate of Compliance
References (21)
ab216653 has been referenced in 21 publications.
- Watanabe T et al. Mesenchymal stem cells attenuate ischemia-reperfusion injury after prolonged cold ischemia in a mouse model of lung transplantation: a preliminary study. Surg Today N/A:N/A (2016). IHC . PubMed: 27484066
- Grau JJ et al. Enrichment of Cells with Cancer Stem Cell-Like Markers in Relapses of Chemoresistant Patients with Locally Advanced Head and Neck Squamous Cell Carcinoma. Oncology 90:267-72 (2016). IHC ; Human . PubMed: 27077749
- Ritz D et al. High-sensitivity HLA class I peptidome analysis enables a precise definition of peptide motifs and the identification of peptides from cell lines and patients' sera. Proteomics N/A:N/A (2016). WB ; Human . PubMed: 26992070
- Lee HJ et al. Prognostic Significance of Tumor-Infiltrating Lymphocytes and the Tertiary Lymphoid Structures in HER2-Positive Breast Cancer Treated With Adjuvant Trastuzumab. Am J Clin Pathol 144:278-88 (2015). PubMed: 26185313
- Mori H et al. Introduction of Zinc-salt Fixation for Effective Detection of Immune Cell-related Markers by Immunohistochemistry. Toxicol Pathol 43:883-9 (2015). PubMed: 26157038