Overview

  • Product name
    Anti-BRN3A antibody [EP1972Y]
    See all BRN3A primary antibodies
  • Description
    Rabbit monoclonal [EP1972Y] to BRN3A
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, Flow Cytmore details
    Unsuitable for: ICC/IF or IP
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human BRN3A aa 300-400 (C terminal). The exact sequence is proprietary.

  • Positive control
    • WB: Human brain lysate; SW480; Mouse cerebrellum; Rat brain; Rat spinal cord. ICC/IF: HeLa cells. FC: SH-SY5Y cells.
  • General notes

     

     

    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.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab81213 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 1/2000. Predicted molecular weight: 43 kDa.

For unpurified use at 1/500 - 1/1000. 

Flow Cyt 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

For unpurified use at 1/90. 

  • Application notes
    Is unsuitable for ICC/IF or IP.
  • Target

    • Function
      Probable transcription factor which may play a role in the regulation of specific gene expression within a subset of neuronal lineages. May play a role in determining or maintaining the identities of a small subset of visual system neurons.
    • Tissue specificity
      Brain. Seems to be specific to the retina. Present in the developing brain, spinal cord and eye.
    • Sequence similarities
      Belongs to the POU transcription factor family. Class-4 subfamily.
      Contains 1 homeobox DNA-binding domain.
      Contains 1 POU-specific domain.
    • Developmental stage
      Expression peaks early in embryogenesis (day 13.5) and is undetectable 14 days after birth.
    • Cellular localization
      Nucleus.
    • Information by UniProt
    • Database links
    • Alternative names
      • Brain specific homeobox/POU domain protein 3A antibody
      • Brain-3A antibody
      • Brain-specific homeobox/POU domain protein 3A antibody
      • Brn 3.0 antibody
      • BRN 3A antibody
      • Brn-3A antibody
      • Brn3 antibody
      • BRN3A antibody
      • class 4 antibody
      • FLJ13449 antibody
      • Homeobox/POU domain protein RDC 1 antibody
      • Homeobox/POU domain protein RDC-1 antibody
      • Homeobox/POU domain protein RDC1 antibody
      • Oct T1 antibody
      • Oct-T1 antibody
      • OctT1 antibody
      • PO4F1_HUMAN antibody
      • POU class 4 homeobox 1 antibody
      • POU domain antibody
      • POU Domain Class 4 Transcription Factor 1 antibody
      • Pou4f1 antibody
      • RDC 1 antibody
      • RDC1 antibody
      • transcription factor 1 antibody
      see all

    Images

    • All lanes : Anti-BRN3A antibody [EP1972Y] (ab81213) at 1/2000 dilution (purified)

      Lane 1 : SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysates
      Lane 2 : Mouse cerebellum lysates
      Lane 3 : Rat brain lysates
      Lane 4 : Rat spinal cord lysates

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 43 kDa



      Blocking and diluting buffer: 5% NFDM/TBST.

    • Flow Cytometry analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells labeling BRN3A with purified ab81213 at 1:100 dilution (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor ® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).
    • All lanes : Anti-BRN3A antibody [EP1972Y] (ab81213) (unpurified)

      Lane 1 : LNCaP
      Lane 2 : SW480
      Lane 3 : PC-3
      Lane 4 : Human brain lysate

      Predicted band size: 43 kDa
      Observed band size: 43 kDa

    • All lanes : Anti-BRN3A antibody [EP1972Y] (ab81213) at 1/2000 dilution (unpurified)

      Lane 1 : Adult mouse cerebellum lysate
      Lane 2 : Adult mouse lower brainstem lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : HRP-conjugated Mouse anti-Rabbit IgG monoclonal. at 1/10000 dilution

      Performed under reducing conditions.

      Predicted band size: 43 kDa
      Observed band size: 43 kDa


      Exposure time: 2 minutes

      See Abreview

    • Overlay histogram showing SH-SY5Y cells stained with unpurified ab81213 (red line). The cells were fixed with 80% methanol (5 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 (ab81213, 1/50 dilution) 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 (monclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

    References

    This product has been referenced in:
    • Wu X  et al. Mitochondria-targeted antioxidant peptide SS-31 mediates neuroprotection in a rat experimental glaucoma model. Acta Biochim Biophys Sin (Shanghai) 51:411-421 (2019). Read more (PubMed: 30811524) »
    • Gong JL  et al. The increased expression of GABA receptors within the arcuate nucleus is associated with high intraocular pressure. Mol Vis 24:574-586 (2018). Read more (PubMed: 30174387) »
    See all 7 Publications for this product

    Customer reviews and Q&As

    1-7 of 7 Abreviews or Q&A

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (Retina)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: 0.01M citrate buffer
    Permeabilization
    Yes - tween 20
    Specification
    Retina
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
    Fixative
    Davidson

    Abcam user community

    Verified customer

    Submitted Nov 05 2018

    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (mouse eye)
    Permeabilization
    Yes - 0.3% triton X-100
    Specification
    mouse eye
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 18°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Mar 18 2016

    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (Retinal ganglion cells)
    Permeabilization
    Yes - Triton-X-100
    Specification
    Retinal ganglion cells
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C
    Fixative
    Paraformaldehyde

    Dr. Kris Pike

    Verified customer

    Submitted Aug 11 2015

    Question

    We used 5% goat blocking serum, and preincubated the sections for 30min, and the wholemount for 2 days. The secondaries have both been used successfully with other primaries over the past 2-3 months. The protocol for the sections is below:



    Slide 1 – Abcam rb α Brn3a 1:100; g α rb Alexa 594 1:400



    Slide 2 – Abcam rb α Brn3a 1:100; g α rb Alexa 488 1:400



    · Let frozen slides air dry for 10min, circle with PAP pen

    · Label slides with pencil (primary and secondary antibody, concentrations and date)


    Wash 3x in PBS (5 min each), coplin jars

    Triton X-100 3% (15 min)

    Wash 3x in PBS (5 min each), coplin jars

    Pre-incubate in 120 µl of 5% Goat Blocking Serum and cover with parafilm for 30 min at RT

    Prepare primary antibodies by adding primary AB to NEW blocking buffer (total volume 100 µl) For double label: need to halve concentration denominator since applying both antibodies together so there is double the volume. Make as a batch for each antibody. Vortex all tubes. Mix double label tubes before applying.




    rb α Brn3a, 1:1 → 1:100 (2/6/2013, Abcam #AB81213)

    2 µl 1:1 + 198 µl GBS

    Gives 200 µl 1:100 total, use 90 µl for each slide: Slide 1, Slide 2




    Pipette 90 µl of the appropriate prepared primary AB onto slides, cover in parafilm for 2 h at RT



    Wash 3x in PBS (5 min each), coplin jars

    Make up secondary antibodies by adding secondary AB to NEW blocking buffer (total volume 100 µl). Use Alexa 594 or 488-coupled antibody at 1:400.




    g α rb Alexa 594 1:1 → 1:400

    2 µl undiluted + 798 µl GBS

    Gives 100 µl 1:400 for Slide 1



    g α rb Alexa 488 1:1 → 1:400

    2 µl undiluted + 798 µl GBS

    Gives 100 µl 1:400 for Slide 2




    Centrifuge antibodies at 12000 G for 5 min

    Incubate sections in secondary antibodies (90 µl) for 1 hour at RT covered with parafilm, keep in dark

    Wash 3x in PBS (15 min each) in dark

    Coverslip on slide with drop of vectashield with DAPI. Keep in dark.

    Read More
    Answer

    With the frozen sections, did you do a fix and permeabilize step. I didnt notice that in your protocol. If you haven't then that 's probably the reason for no staining. Its important to do that so that the epitope is accessible.
    I would also just incubate the primary antibody at 4 degrees overnight.

    Read More
    This product is known to not work in this application or species.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (Dorsal Root Ganglia (DRG) neurons)
    Specification
    Dorsal Root Ganglia (DRG) neurons
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - tween 20
    Blocking step
    Serum as blocking agent for 30 minute(s) · Concentration: 2% · Temperature: 21°C

    Abcam user community

    Verified customer

    Submitted Oct 18 2012

    Application
    IHC - Wholemount
    Sample
    Mouse Tissue (Brain explant)
    Specification
    Brain explant

    Abcam user community

    Verified customer

    Submitted Oct 05 2012

    Application
    Western blot
    Sample
    Mouse Tissue lysate - whole (cerebellum & lower portion of brainstem (adult))
    Loading amount
    20 µg
    Specification
    cerebellum & lower portion of brainstem (adult)
    Gel Running Conditions
    Reduced Denaturing (12 %)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

    Abcam user community

    Verified customer

    Submitted Feb 15 2011

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