Recombinant Anti-AICDA antibody [EPR23436-45] - ChIP Grade (ab269454)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR23436-45] to AICDA - ChIP Grade
- Suitable for: Flow Cyt (Intra), IHC-P, ICC/IF, IP, WB, ChIP
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-AICDA antibody [EPR23436-45] - ChIP Grade
See all AICDA primary antibodies -
Description
Rabbit monoclonal [EPR23436-45] to AICDA - ChIP Grade -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt (Intra), IHC-P, ICC/IF, IP, WB, ChIPmore details -
Species reactivity
Reacts with: Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Ramos,NAMALWA and Raji lysates. IHC-P: Human tonsil, Human Hodgkin lymphoma and Human diffuse large B-cell lymphoma tissues. ICC/IF: NAMALWA cells. Flow Cyt (intra): NAMALWA cells. IP: Ramos and NAMALWA cells.ChIP: Chromatin prepared from Ramos cells.
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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
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 short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR23436-45 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab269454 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) |
1/500.
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IHC-P | (1) |
1/4000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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ICC/IF |
1/50.
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IP |
1/30.
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WB |
1/1000. Detects a band of approximately 24 kDa (predicted molecular weight: 23 kDa).
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ChIP |
Use 5 µg for 25 µg of chromatin.
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Notes |
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Flow Cyt (Intra)
1/500. |
IHC-P
1/4000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
ICC/IF
1/50. |
IP
1/30. |
WB
1/1000. Detects a band of approximately 24 kDa (predicted molecular weight: 23 kDa). |
ChIP
Use 5 µg for 25 µg of chromatin. |
Target
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Function
RNA-editing deaminase involved in somatic hypermutation, gene conversion, and class-switch recombination. Required for several crucial steps of B-cell terminal differentiation necessary for efficient antibody responses. -
Tissue specificity
Strongly expressed in lymph nodes and tonsils. -
Involvement in disease
Defects in AICDA are the cause of hyper-IgM immunodeficiency syndrome type 2 (HIGM2) [MIM:605258]; also known as hyper-IgM syndrome 2. HIGM2 is an autosomal recessive disorder characterized by normal or elevated serum IgM levels with absence of IgG, IgA, and IgE, resulting in a profound susceptibility to bacterial infections. HIGM2 causes the absence of Ig class switch recombination (CSR), the lack of Ig somatic hypermutations, and lymph node hyperplasia caused by the presence of giant germinal centers. -
Sequence similarities
Belongs to the cytidine and deoxycytidylate deaminase family. - Information by UniProt
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Database links
- Entrez Gene: 57379 Human
- Omim: 605257 Human
- SwissProt: Q9GZX7 Human
- Unigene: 149342 Human
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Alternative names
- Activation induced cytidine deaminase antibody
- Activation induced deaminase antibody
- Activation-induced cytidine deaminase antibody
see all
Images
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All lanes : Anti-AICDA antibody [EPR23436-45] - ChIP Grade (ab269454) at 1/1000 dilution
Lane 1 : Wild-type Raji cell lysate
Lane 2 : AICDA knockout Raji cell lysate
Lane 3 : Daudi cell lysate
Lane 4 : SH-SY5Y cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 23 kDa
Observed band size: 22 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-AICDA antibody [EPR23436-45] - ChIP Grade staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab269454 was shown to bind specifically to AICDA. A band was observed at 22 kDa in wild-type Raji cell lysates with no signal observed at this size in AICDA knockout cell line ab277185 (knockout cell lysate ab277227). To generate this image, wild-type and AICDA knockout Raji 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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Chromatin was prepared from Ramos cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30 mins and then formaldehyde for 10 min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab269454 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are commercial primers from paper: PMC2905439
*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol
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Immunohistochemical analysis of paraffin-embedded Human diffuse large B-cell lymphoma tissue labeling AICDA with ab269454 at 1/4000 dilution (0.12ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mainly cytoplasmic staining (weak nuclear staining) in part of tumor cells of human diffuse large B-cell lymphoma (PMID: 29251015).
The section was incubated with ab255611 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.
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Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NAMALWA (human Burkitt's lymphoma B lymphocyte) and K-562 (human chronic myelogenous leukemia lymphoblast) cells labelling AICDA with ab269454 at 1/50 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in NAMALWA cell line. Negative control: K-562 cell line (PMID: 27217538). ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).
Secondary antibody only control: Secondary antibody is ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.
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Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized Ramos (human Burkitt's lymphoma B lymphocyte) cells labelling AICDA with ab269454 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.
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Immunohistochemical analysis of paraffin-embedded Human Hodgkin lymphoma tissue labeling AICDA with ab269454 at 1/4000 dilution (0.12ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mainly cytoplasmic staining (weak nuclear staining) in part of tumor cells of human Hodgkin lymphoma (PMID: 15732141).
The section was incubated with ab255611 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.
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Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling AICDA with ab269454 at 1/4000 dilution (0.12ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Mainly cytoplasmic staining (weak nuclear staining) in germinal center cells of human tonsil (PMID:23877718, 15732141, PMID: 29251015).
The section was incubated with ab255611 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.
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AICDA was immunoprecipitated from 0.35 mg NAMALWA (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab269454 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab269454 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.
Lane 1: NAMALWA whole cell lysate 10ug.
Lane 2: ab269454 IP in NAMALWA whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab269454 in NAMALWA whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 90 seconds.
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AICDA was immunoprecipitated from 0.35 mg Ramos (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab269454 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab269454 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.
Lane 1: Ramos whole cell lysate 10ug.
Lane 2: ab269454 IP in Ramos whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab269454 in Ramos whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 90 seconds.
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Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized K-562 (human chronic myelogenous leukemia lymphoblast, Left) / NAMALWA (human Burkitt's lymphoma B lymphocyte, Right) cells labelling AICDA with ab269454 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.
Negative control: K-562 cell line (PMID: 27217538).
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All lanes : Anti-AICDA antibody [EPR23436-45] - ChIP Grade (ab269454) at 1/1000 dilution
Lane 1 : Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate
Lane 2 : K-562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate
Lane 3 : NAMALWA (human Burkitt's lymphoma B lymphocyte), whole cell lysate
Lane 4 : Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Predicted band size: 23 kDa
Observed band size: 24 kDa why is the actual band size different from the predicted?Blocking and dilution buffer: 5% NFDM/TBST.
Exposure time: 114 seconds.
Negative control: K-562 (PMID: 27217538).
The expression profile observed is consistent with what has been described in the literature (PMID: 27217538).
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.
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (0)
ab269454 has not yet been referenced specifically in any publications.