Recombinant Anti-CXCR5 antibody [EPR23463-30] (ab254415)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR23463-30] to CXCR5
- Suitable for: ICC/IF, WB, Flow Cyt, IHC-P
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-CXCR5 antibody [EPR23463-30]
See all CXCR5 primary antibodies -
Description
Rabbit monoclonal [EPR23463-30] to CXCR5 -
Host species
Rabbit -
Specificity
We observe only weak staining in human WB. We do not suggest this product for use in IHC with mouse or rat.
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Tested applications
Suitable for: ICC/IF, WB, Flow Cyt, IHC-Pmore details
Unsuitable for: IP -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: Raji, Daudi, Neuro-2a, A20, Mouse spleen, mouse lymph node and rat lymph node, C6 lysates. IHC-P: Human tonsil tissue. Human diffuse large B- lymphoma tissue. Flow Cyt: Human peripheral blood mononuclear and Raji cells. ICC/IF: Daudi 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
EPR23463-30 -
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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KO cell lines
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KO cell lysates
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab254415 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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ICC/IF |
Use a concentration of 5 µg/ml.
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WB |
1/1000. Predicted molecular weight: 42 kDa.
We observe only weak staining in human WB. |
|
Flow Cyt | (1) |
1/500.
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IHC-P |
1/5000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
We do not suggest this product for use in IHC with mouse or rat. |
Notes |
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ICC/IF
Use a concentration of 5 µg/ml. |
WB
1/1000. Predicted molecular weight: 42 kDa. We observe only weak staining in human WB. |
Flow Cyt
1/500. |
IHC-P
1/5000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. We do not suggest this product for use in IHC with mouse or rat. |
Target
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Function
Cytokine receptor that binds to B-lymphocyte chemoattractant (BLC). Involved in B-cell migration into B-cell follicles of spleen and Peyer patches but not into those of mesenteric or peripheral lymph nodes. May have a regulatory function in Burkitt lymphoma (BL) lymphomagenesis and/or B-cell differentiation. -
Tissue specificity
Expression in mature B-cells and Burkitt lymphoma cells. -
Sequence similarities
Belongs to the G-protein coupled receptor 1 family. -
Cellular localization
Cell membrane. - Information by UniProt
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Database links
- Entrez Gene: 643 Human
- Entrez Gene: 12145 Mouse
- Entrez Gene: 29363 Rat
- Omim: 601613 Human
- SwissProt: P32302 Human
- SwissProt: Q04683 Mouse
- SwissProt: P34997 Rat
- Unigene: 113916 Human
see all -
Alternative names
- BLR 1 antibody
- BLR1 antibody
- Burkitt lymphoma receptor 1 antibody
see all
Images
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Flow cytometry overlay histogram showing wild-type Raji (green line) and CXCR5 knockout Raji cells (ab273380) stained with ab254415 (red line). The cells were incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serumto block FC receptors and non-specific protein-protein interaction followed by the antibody (ab254415) (1x106 in 100μl at 0.2 μg/ml) for 30 min at 4°C.
The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 4°C.
Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type Raji - black line; CXCR5 knockout Raji - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
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Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CXCR5 with ab254415 at 1/5000 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human tonsil (PMID: 12393412). The section was incubated with ab254415 for 10 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 ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.
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All lanes : Anti-CXCR5 antibody [EPR23463-30] (ab254415) at 1/1000 dilution
Lane 1 : Wild-type Raji cell lysate
Lane 2 : CXCR5 knockout Raji cell lysate
Lane 3 : Daudi cell lysate
Lane 4 : Jurkat cell lysate
Lysates/proteins at 30 µg per lane.
Performed under reducing conditions.
Predicted band size: 42 kDaLanes 1 - 4: Merged signal (red and green). Green - ab254415 observed at 60 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.
ab254415 was shown to react with CXCR5 in Raji wild-type cells in Western blot with loss of signal observed in CXCR5 knockout sample. Wild-type and CXCR5 knockout Raji cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab254415 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 h at room temperature before imaging.
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ab254415 staining CXCR5 in Daudi cells (top panel, positive control) and Jurkat cells (bottom panel, negative control). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab254415 at 5μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Flow cytometric analysis of Raji (Human Burkitt's lymphoma B lymphocyte) cells labelling CXCR5 with ab254415 at 1/500 dilution (0.1µg) (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). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.
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Flow cytometric analysis of human peripheral blood mononuclear cell (PBMC) cells labelling CXCR5 with ab254415 at 1/500 dilution (0.1µg) (Right) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary at a 1/2000 dilution. Cells were stained with rabbit IgG (Left) or ab254415 (Right), then stained with anti-CD19 conjugated to Alexa Fluor® 647.
Gated on viable cells.
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All lanes : Anti-CXCR5 antibody [EPR23463-30] (ab254415) at 1/1000 dilution
Lane 1 : Neuro-2a (mouse neuroblastoma neuroblast), whole cell lysate
Lane 2 : A20 (mouse reticulum sarcoma B lymphocyte), whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution
Predicted band size: 42 kDa
Observed band size: 42 kDaBlocking and diluting buffer and concentration: 5% NFDM/TBST.
Samples are non-boiled as boiling may cause protein aggregates.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 30553016).
Exposure time: 20 seconds.
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All lanes : Anti-CXCR5 antibody [EPR23463-30] (ab254415) at 1/1000 dilution
Lane 1 : Rat lymph node tissue lysate
Lane 2 : C6 (rat glial tumor glial cell), whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution
Predicted band size: 42 kDa
Observed band size: 42 kDaBlocking and diluting buffer and concentration: 5% NFDM/TBST.
This blot was developed using a higher sensitivity ECL substrate.
Samples are non-boiled as boiling may cause protein aggregates.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 30553016).
Exposure time: 122 seconds.
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All lanes : Anti-CXCR5 antibody [EPR23463-30] (ab254415) at 1/1000 dilution
Lane 1 : Mouse spleen tissue lysate
Lane 2 : Mouse lymph node tissue lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution
Predicted band size: 42 kDa
Observed band size: 42 kDaBlocking and diluting buffer and concentration: 5% NFDM/TBST.
This blot was developed using a higher sensitivity ECL substrate.
Samples are non-boiled as boiling may cause protein aggregates.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 30553016).
Exposure time: 122 seconds.
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Immunohistochemical analysis of paraffin-embedded human diffuse large B-cell lymphoma tissue labeling CXCR5 with ab254415 at 1/5000 dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human diffuse large B-cell lymphoma. The section was incubated with ab254415 for 10 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 ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.
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 (1)
ab254415 has been referenced in 1 publication.
- Lou W et al. Bromodomain-containing protein 9 is a prognostic biomarker associated with immune infiltrates and promotes tumor malignancy through activating notch signaling pathway in negative HIF-2α clear cell renal cell carcinoma. IUBMB Life 73:1334-1347 (2021). PubMed: 34415102