Overview

  • Product name
  • Description
    Goat polyclonal to Iba1
  • Host species
    Goat
  • Specificity
    This antibody is expected to recognise isoform 3 (NP_001614.3) only. This antibody gave excellent staining in paraffin embedded human adult tissue and embyronic rat tissue. For WB, we recommend blocking in 3-5% milk, NOT BSA. Blocking in BSA gives high background.
  • Tested applications
    Suitable for: WB, IHC-Fr, IHC-Pmore details
    Unsuitable for: IHC-FoFr
  • Species reactivity
    Reacts with: Mouse, Rabbit, Human
    Predicted to work with: Cow, Pig, Macaque monkey
  • Immunogen

    Synthetic peptide corresponding to Human Iba1 aa 28-42 (internal sequence) (Cysteine residue). NP_001614.3
    Sequence:

    C-NKQFLDDPKYSSDED


    Database link: P55008

  • Positive control
    • WB: Recombinant Human Iba1 protein (ab117478), Human peripheral blood mononucleocyte and frontal cortex lysate. IHC-P: Mouse brain; Human placenta and jejunum; rat embryonic liver tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab48004 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 Use a concentration of 0.5 - 1.5 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 17 kDa).

We recommend blocking in 3-5% milk, not BSA. Blocking in BSA gives high background in WB.

IHC-Fr Use at an assay dependent concentration.
IHC-P Use a concentration of 2 - 4 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
  • Application notes
    Is unsuitable for IHC-FoFr.
  • Target

    • Function
      Actin-binding protein that enhances membrane ruffling and RAC activation. Enhances the actin-bundling activity of LCP1. Binds calcium. Plays a role in RAC signaling and in phagocytosis. May play a role in macrophage activation and function. Promotes the proliferation of vascular smooth muscle cells and of T-lymphocytes. Enhances lymphocyte migration. Plays a role in vascular inflammation.
    • Tissue specificity
      Detected in T-lymphocytes and peripheral blood mononuclear cells.
    • Sequence similarities
      Contains 2 EF-hand domains.
    • Post-translational
      modifications
      Phosphorylated on serine residues.
    • Cellular localization
      Cytoplasm > cytoskeleton. Cell projection > ruffle membrane. Associated with the actin cytoskeleton at membrane ruffles and at sites of phagocytosis.
    • Information by UniProt
    • Database links
    • Alternative names
      • AIF 1 antibody
      • AIF-1 antibody
      • Aif1 antibody
      • AIF1 protein antibody
      • AIF1_HUMAN antibody
      • Allograft inflammatory factor 1 antibody
      • Allograft inflammatory factor 1 splice variant G antibody
      • allograft inflammatory factor-1 splice variant Hara-1 antibody
      • balloon angioplasty responsive transcription antibody
      • BART 1 antibody
      • G1 antibody
      • G1 putative splice variant of allograft inflamatory factor 1 antibody
      • IBA 1 antibody
      • IBA1 antibody
      • interferon gamma responsive transcript antibody
      • Interferon responsive transcript 1 antibody
      • interferon responsive transcript factor 1 antibody
      • Ionized calcium binding adapter molecule 1 antibody
      • Ionized calcium-binding adapter molecule 1 antibody
      • ionized calcium-binding adapter molecule antibody
      • IRT 1 antibody
      • IRT1 antibody
      • Microglia response factor antibody
      • MRF1 antibody
      • Protein g1 antibody
      see all

    Images

    • Paraffin embedded mouse brain tissue stained for Iba1 with ab48004 (2 µg/ml) in immunohistochemical analysis. Steamed antigen retrieval with citrate buffer pH 6, HRP-staining.

    • Anti-Iba1 antibody (ab48004) at 0.5 µg/ml + human frontal cortex lysate in RIPA buffer at 35 µg

      Developed using the ECL technique.

      Predicted band size: 17 kDa



      Primary incubation was 1 hour

    • Paraformaldehyde fixed mouse brain tissue stained for Iba1 with ab48004 (1/200 dilution) in immunohistochemical analysis. Secondary: Donkey Anti-Goat IgG Cy3® (1/500 dilution).

      See Abreview

    • All lanes : Anti-Iba1 antibody (ab48004) at 1 µg/ml

      Lane 1 : Human peripheral blood mononucleocyte lysate in RIPA buffer
      Lane 2 : Human peripheral blood mononucleocyte lysate in RIPA buffer with blocking peptide

      Lysates/proteins at 35 µg per lane.

      Developed using the ECL technique.

      Predicted band size: 17 kDa
      Observed band size: 17 kDa
      Additional bands at: 110 kDa, 35 kDa. We are unsure as to the identity of these extra bands.



      1 hour primary antibody incubation. Additional bands of unknown identity observed at 35kDa and 110kDa. These bands were successfully blocked by incubation with the immunising peptide
    • ab48004 at 1/300 staining human jejunum tissue sections by IHC-P. A heat mediated antigen retrieval step in citric acid buffer was performed and the tissue was blocked in BSA before incubation with the antibody for 2 hours. A biotinylated rabbit anti-goat antibody was used as the secondary.

      See Abreview

    • ab48004 diluted 1/200 with TBS/BSA/Azide staining rat d15 embryonic liver tissue sections by IHC-P.  The tissue was formaldehyde fixed and a heat-mediated antigen retrieval step in citric acid buffer at pH6 was performed prior to blocking with 1% BSA for 10 minutes at room temperature.  Incubation time with the primary antibody was 16 minutes.  A biotinylated rabbit anti-goat was used as the secondary.

      See Abreview

    • Paraffin embedded human placenta tissue stained for Iba1 with ab48004 (2.5 µg/ml) in immunohistochemical analysis. A heat mediated antigen retrieval step in citric acid buffer (pH 6) was performed.

    References

    This product has been referenced in:
    • Wen J  et al. WWL70 protects against chronic constriction injury-induced neuropathic pain in mice by cannabinoid receptor-independent mechanisms. J Neuroinflammation 15:9 (2018). Mouse . Read more (PubMed: 29310667) »
    • Kim MJ  et al. The Anti-Inflammatory Effects of Oral-Formulated Tacrolimus in Mice with Experimental Autoimmune Encephalomyelitis. J Korean Med Sci 32:1502-1507 (2017). Read more (PubMed: 28776347) »
    See all 6 Publications for this product

    Customer reviews and Q&As

    1-10 of 14 Abreviews or Q&A

    Abcam has not validated the combination of species/application used in this Abreview.
    Application
    Immunohistochemistry free floating
    Sample
    Mouse Tissue sections (cortex)
    Specification
    cortex

    Abcam user community

    Verified customer

    Submitted Jun 15 2018

    Abcam has not validated the combination of species/application used in this Abreview.
    Application
    Immunohistochemistry (PFA perfusion fixed frozen sections)
    Sample
    Mouse Tissue sections (spinal cord)
    Antigen retrieval step
    None
    Permeabilization
    Yes - 0.3% TritonX-100
    Specification
    spinal cord
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
    Fixative
    Paraformaldehyde

    Jennifer Deberry

    Verified customer

    Submitted Mar 15 2016

    Answer

    Thank you for your enquiry regarding ab48004 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody.

    Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.

    As the datasheet indicates under the Specificity section, this antibody is expected to recognise isoform 3 (NP_001614.3) only. It gave excellent staining in paraffin embedded human adult tissue and embyronic rat tissue. However, adult rat/mouse brain microglia and adult/embryonic mouse non-neuronal macrophages staining was very faint compared to another Iba1 antibody.

    1) Main questions: Are the samples from rat spinal cord from adult, if so at what age? This seems to be an important piece of information.

    2) What percentage normal Donkey serum was used for blocking? At what temperature and for how long?

    3) Does the detection system work fine? Have you used it successfully with another primary antibody?

    4) What positive control was applied? Human Peripheral Blood Mononucleocyte lysate

    5) Has the customer got any preliminary data or specific published papers to confirm that chronic constriction of the sciatic nerve could induce Iba1 expression? Could you please explain and specify the paper you are referring to (by Benett and Xie)?

    I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

    Read More

    Answer

    Thank you for your reply.

    Your protocol seems comprehensive, although I would strongly recommend including the antigen retrieval step since this has worked well for other customers. We would recommend citrate buffer pH 6, which we do sell as ab64214.

    https://www.abcam.com/10x-citrate-buffer-ph-6-0-ab64214.html

    If you're looking for an antigen retrieval protocol, we have some online as well.

    https://www.abcam.com/index.html?pageconfig=resource&rid=11488

    If you do try the antigen retrieval step and still see no staining, I would be happy to replace or refund the product for you.

    I hope this information helps. Please contact us with any other questions.

    Read More

    Answer

    Thank you for contacting us.

    I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

    Would you be able to provide more information about the protocol that was tested? The antibody is guaranteed to work in rabbit, so if we can't figure out why it's not working we'd be happy to offer a replacement or refund.

    Were the tissues paraffin-embedded (IHC-P) or frozen (IHC-Fr)? How were they fixed?

    This is a cytoplasmic protein, so was a permeabilization step included? If so, with which agent?

    Did you perform antigen retrieval? If so, with what buffer and how long?

    How long was the antibody incubated? We would generally recommend overnight at 4C.

    What type of rabbit tissue was stained (i.e organ)? Did you run a positive control with human jejunum or placenta? The data we have for rabbit comes from brain and can be seen here: https://www.abcam.com/index.html?datasheet=48004&tab=abreviews&intabreviewid=18504

    I look forward to receiving your reply so that we can help you further with this antibody.

    Read More
    Application
    Immunohistochemistry (PFA perfusion fixed frozen sections)
    Sample
    Rat Tissue sections (Brain)
    Specification
    Brain
    Fixative
    Formaldehyde
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Citrate buffer
    Permeabilization
    Yes - 0.1% TritonX-100
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted Jul 13 2012

    Answer

    Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

    The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

    I would like to reassure you that ab48004 is tested and covered by our 6 month guarantee for use inWB and human samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

    Reviewing this case, I would like to offer some suggestions to help optimise the results from ab48004.

    1.) Since there is no band, I can recommend to not use any blocking agent in the antibody solution. I also do not recommend to mix blocking agents and suggest to use BSA for blocking.

    2.) I suggest to incubate the primary antibody over night at 4C to achieve full saturation.

    3.) Maybe a more standard procedure in developing will give better results. maybe some form of ECL kit will improve the results.

    4.) I can also suggest to use SDS in the lysis buffer to achieve complete recovery of the protein and to boil the samples for 10min before loading them on the gel.

    I would also appreciate if you can confirm some further details:

    Is this method working with other primary antibodies in your lab?

    Is the secondary antibody functional?

    Are there any bands on the blot and is the loading control working?

    I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

    Read More
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Rabbit Tissue sections (brain)
    Specification
    brain
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - triton (20 ul in 10 ml PBS)
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Dr. Praveen Ballabh

    Verified customer

    Submitted Jul 22 2010

    Abcam has not validated the combination of species/application used in this Abreview.
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Dog Tissue sections (spinal cord)
    Specification
    spinal cord
    Fixative
    formalin
    Permeabilization
    Yes - 0.3% PBS-TX
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

    Joanne Steinauer

    Verified customer

    Submitted Jun 08 2010

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (brain)
    Specification
    brain
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.5%TritonX-100/PBS
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted Jul 15 2009

    1-10 of 14 Abreviews or Q&A

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