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Anti-Spastin抗体[Sp 3G11/1] (ab31850)

Submit a review Q&A (4)References (5)
Western blot - Anti-Spastin antibody [Sp 3G11/1] (ab31850)
  • Immunoprecipitation - Anti-Spastin antibody [Sp 3G11/1] (ab31850)
  • Western blot - Anti-Spastin antibody [Sp 3G11/1] (ab31850)
  • Western blot - Anti-Spastin antibody [Sp 3G11/1] (ab31850)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Spastin antibody [Sp 3G11/1] (ab31850)

Key features and details

  • Mouse monoclonal [Sp 3G11/1] to Spastin
  • Suitable for: WB, IHC-P, IP
  • Knockout validated
  • Reacts with: Rat, Human
  • Isotype: IgG2a

概述

  • 产品名称

    Anti-Spastin抗体[Sp 3G11/1]
    参阅全部 Spastin 一抗

  • 描述

    小鼠单克隆抗体[Sp 3G11/1] to Spastin

  • 宿主

    Mouse

  • 经测试应用

    适用于: WB, IHC-P, IPmore details

  • 种属反应性

    与反应: Rat, Human

  • 免疫原

    Recombinant full length protein (Human).

  • 阳性对照

    • WB: Total HeLa extract or rat brain synaptosome This antibody gave a positive result in IHC in the following FFPE tissue: Human lung adenocarcinoma. IP: HAP1 cells

  • 常规说明

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

性能

应用

The Abpromise guarantee

Abpromise™承诺保证使用ab31850于以下的经测试应用

“应用说明”部分 下显示的仅为推荐的起始稀释度；实际最佳的稀释度/浓度应由使用者检定。

应用 Ab评论 说明
WB
1/500. Detects a band of approximately 52 kDa.

There are two splice isoforms of spastin, one without exon4 and two alternative ATG start sites, which may determine the localisation of the translate protein. 52kDa is the major band.
IHC-P
Use at an assay dependent concentration.
IP
Use at an assay dependent concentration.
说明
WB
1/500. Detects a band of approximately 52 kDa.

There are two splice isoforms of spastin, one without exon4 and two alternative ATG start sites, which may determine the localisation of the translate protein. 52kDa is the major band.
IHC-P
Use at an assay dependent concentration.
IP
Use at an assay dependent concentration.

靶标

  • 相关性

    Spastin is thought have a role in microtubule dynamics through its function as a microtubule severing protein. It is localised to the centrosome of neuronal cells but is not found in glial cells. Mutation in the ATPase binding domain of spastin causes hereditary spastic paraplegias (HSP), a large group of clinically similar disorders. Mutant forms of spastin are generally found throughout the cytoplasm rather then within the nucleus.

  • 细胞定位

    Cytoplasm, Cytoskeleton, Endoplasmic reticulum, Endosome, Membrane, Microtubule, Nucleus

  • 数据库链接

    • 别名

      • ADPSP antibody
      • FSP 2 antibody
      • FSP2 antibody
      • KIAA1083 antibody
      • Spast antibody
      • spastic paraplegia 4 (autosomal dominant; spastin) antibody
      • Spastic paraplegia 4 antibody
      • spastin antibody
      • SPG 4 antibody
      • SPG4 antibody
      • SPG4 gene antibody
      see all

    图片

    • Western blot - Anti-Spastin antibody [Sp 3G11/1] (ab31850)
      Western blot - Anti-Spastin antibody [Sp 3G11/1] (ab31850)
      All lanes : Anti-Spastin antibody [Sp 3G11/1] (ab31850) at 1/500 dilution

      Lane 1 : Wild-type HEK-293T cell lysate
      Lane 2 : SPAST knockout HEK293T cell lysate
      Lane 3 : Wild-type HAP1 cell lysate
      Lane 4 : SPAST knockout HAP1 cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : goat anti-rabbit HRP at 0.2 µg/ml

      Performed under reducing conditions.


      ab31850 was shown to react with SPAST in wild-type HEK293T cells in Western blot with loss of signal observed in SPAST knockout cell line ab267238 (SPAST knockout cell lysate ab258698). Wild-type HEK293T and SPAST knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab31850 overnight at 4 °C at a 1/500 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 0.2μg/mL before imaging.

      These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

    • Immunoprecipitation - Anti-Spastin antibody [Sp 3G11/1] (ab31850)
      Immunoprecipitation - Anti-Spastin antibody [Sp 3G11/1] (ab31850)

      Immunoprecipitation of SPAST in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 1µg of ab31850 pre-coupled to Protein A beads. Samples were washed and processed for western blot with ab77144 at 1/500.

      This data was kindly provided by the YCharOS Inc., an open science company with the mission of characterizing every commercially available antibody reagent. Abcam are working with YCharOS to support their mission of antibody characterisation using knock out cell lines.

    • Western blot - Anti-Spastin antibody [Sp 3G11/1] (ab31850)
      Western blot - Anti-Spastin antibody [Sp 3G11/1] (ab31850)
      All lanes : Anti-Spastin antibody [Sp 3G11/1] (ab31850) at 1/500 dilution

      Lane 1 : Wild-type HEK-293T cell lysate
      Lane 2 : Spastin knockout HEK-293T cell lysate
      Lane 3 : SH-SY5Y cell lysate
      Lane 4 : HeLa cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Observed band size: 55 kDa why is the actual band size different from the predicted?



      False colour image of Western blot: Anti-Spastin antibody [Sp 3G11/1] staining at 1/500 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab31850 was shown to bind specifically to Spastin. A band was observed at 55 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in SPAST knockout cell line ab267238 (knockout cell lysate ab258698). To generate this image, wild-type and SPAST knockout HEK-293T 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

    • Western blot - Anti-Spastin antibody [Sp 3G11/1] (ab31850)
      Western blot - Anti-Spastin antibody [Sp 3G11/1] (ab31850)
      Anti-Spastin antibody [Sp 3G11/1] (ab31850) at 1/500 dilution + rat brain synaptosome

      Observed band size: 52 kDa why is the actual band size different from the predicted?



      There are two splice isoforms of spastin (one without exon4) and two alternative ATG start sites, which may determine the localisation of the translate protein. 52kDa is the major band.
    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Spastin antibody [Sp 3G11/1] (ab31850)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Spastin antibody [Sp 3G11/1] (ab31850)

      IHC image of Spastin staining in Human lung adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab31850, 10µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

      For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    文献 (5)

    发表研究结果有使用 ab31850？请让我们知道，以便我们可以引用本数据表中的参考文章。

    ab31850 被引用在 5 文献中.

    • Cheers SR  et al. Spastin is an essential regulator of male meiosis, acrosome formation, manchette structure and nuclear integrity. Development 150:N/A (2023). PubMed: 36971361
    • Shen W  et al. Spastin is required for human immunodeficiency virus-1 efficient replication through cooperation with the endosomal sorting complex required for transport (ESCRT) protein. Virol Sin 38:448-458 (2023). PubMed: 37172824
    • Nicholson JM  et al. Chromosome mis-segregation and cytokinesis failure in trisomic human cells. Elife 4:N/A (2015). PubMed: 25942454
    • Daftary GS  et al. A Novel Role for the AAA ATPase Spastin as a HOXA10 Transcriptional Corepressor in Ishikawa Endometrial Cells. Mol Endocrinol 25:1539-49 (2011). WB, ICC/IF, IHC-P, ChIP ; Human . PubMed: 21757506
    • Salinas S  et al. Human spastin has multiple microtubule-related functions. J Neurochem 95:1411-20 (2005). PubMed: 16219033

    客户评价及客户问答

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    1-4 of 4 Abreviews or Q&A

    Question

    Thank you again for your reply. Below are the answers to your questions.




    Spastin ab31850

    Please see attached blot image.



    GPSM2 antibody – It would be great if we could get a replacement to try.


    FIGN ab122238

    We have tried fixation in both Methanol and PFA as well and are unable to detect any protein in any conditions by IHC-P. As well, we have done multiple ECL exposure times and tested additional secondary antibody concentrations with no luck. Do you have any other suggestions?

    Read More
    Answer

    Thank you for your response.

    I would expect ab31850 to give clearer results in WB, even once the many isoforms are taken into consideration. I would be happy to offer you a free of charge replacement, credit, or refund for this antibody as well. Please let me know which antibodies you would like to receive as replacements and I will be happy to process your request.


    For the FIGN antibody ab122238, I would recommend ensuring that you are performing heat mediated antigen retrieval with Citrate buffer, pH 6. In order to increase the signal, it could help to lengthen your antigen retrieval time. Can you please let me know what type of tissue sections you were testing?

    Read More

    Question

    Thank you for getting back to me. Sorry for the delay in getting this information to you. We have actually been testing a third antibody from you that is also giving us the same problem - anti–GPSM2 antibody (a b84571). Hopefully,
    you will be able to assist us.
    Below is the information you requested. I've included the information as well for the anti-GPSM2 antibody.

    1) What was your original order or PO number?

    Anti-spastin antibody (ab31850) - ***
    Anti-FIGN antibody (ab122238) - ***
    Anti-GPSM2 antibody (ab84571) - ***

    2) Could you please provide a description of the problem (no signal, high background, incorrectstaining, etc) or an image of your results?
    SPASTIN – many bands of incorrect size. None of the observed bands disappeared or decreased in intensity in cells transfected with siRNAs against Spastin.
    FIGN – no signal
    GPSM2 – no signal
    The mRNAs for all these genes were detected by RT-PCR in the cells that were used for the WB.

    3) What type of samples were you testing?
    Which species were they from, what tissue type or cell line, and how were they prepared?
    For example, what lysis buffer was used in WB, were the samples reduced and denatured, and how much protein was loaded?
    For IF, how were the cells fixed and permeabilized?
    Proteins extracts from RPE-1 (human), HeLa (human) and NIH3T3 (mouse) were used to test the FIGN and SPASTIN antibodies.
    Proteins extracts from HeLa (human) were used to test the GPSM2 antibody.
    The protein extracts were prepared with the following lysis buffer: 50mM HEPES, 100mM KCl, 0.3% NP-40, 10% glycerol, 1mM EGTA, 1mM MgCl2 + protease inhibitors and
    DTT.
    The protein extracts were quantified by the Bradford assay and 25mg were loaded/lane in 10% SDS-PAGE after
    being incubated at 95ºC for 5 min.

    4) What were the dilutions and incubation times for the block, primary, and secondary antibodies?
    What blocking agent and secondary antibody were used?
    The proteins were transferred to PVDF membranes. The membranes were blocked for 1h in 5% Skimmilk in TBS-Tween. The membranes were incubated with the primary antibodies in
    blocking solution over night at 4ºC with constant agitation. For each antibody the following dilutions were tested: 1:100; 1:250; 1:500; 1.1000.
    After the incubation with the primary antibodies the membranes were washed 3 times for 10 minutes with TBS-Tween. The membranes were then incubated with HRP- conjugated secondary
    antibodies for 1h at room temperature. After this incubation the membranes were washed 3 times with TBS-Tween. Finally the membranes were incubated with ECL and exposed.

    If you require any additional information please let me know. Any help you can provide would be great.

    Read More
    Answer

    Thank you for your reply.

    For the Spastin antibody ab31850, could you please provide an image of your blot or let me know what size bands you are observing? Spastin has several known isoforms that could account for multiple bands between 54-67 kDa. Regarding the lack of change with transfected cells and siRNA, can you tell me if the transfected Spastin was full length and from human? What siRNA were you using, and was the knock-down validated by RT-PCR?

    For the GPSM2 antibody, the protocol you provided seems fine and we would definitely expect you to get good signal using this method in HeLa cells by WB and ICC/IF. I would be happy to offer you a free of charge replacement, credit, or refund for this antibody.

    The FIGN antibody ab122238 has not been validated for use in WB or ICC/IF on cultured cells. Have you tried this antibody in IHC-P? It is possible that it may work in WB or ICC/IF, but we do not have any information about how the antibody would work for those applications. To increase your signal, you could try preparing your samples with RIPA buffer, which is more effective at releasing nuclear proteins. You could also try increasing your ECL exposure time or your secondary antibody concentration.

    I hope this helps, if not, please let me know and I will be happy to help you further.

    Read More

    Question

    We recently ordered two antibodies from you.


    anti-spastin antibody, cat# ab31850

    Anti-FIGN antibody, cat# ab122238


    We have been testing both antibodies by WB and IF. By both WB and IF, both antibodies do not work. Is it possible to test a replacement or get a refund.

    Read More
    Answer

    Thank you for contacting us. I am sorry that these antibodies are not working in WB and IF. We will be happy to refund or replace the antibodies if they are being used in a tested species and application, and have been purchased in the past six months. In order to help our QC process, could you please provide some additional information?

    1) What was your original order or PO number?

    2) Could you please provide a description of the problem (no signal, high background, incorrect staining, etc) or an image of your results?

    3) What type of samples were you testing? Which species were they from, what tissue type or cell line, and how were they prepared? For example, what lysis buffer was used in WB, were the samples reduced and denatured, andhow much protein was loaded? For IF, how were the cells fixed and permeabilized?

    4) What were the dilutions and incubation times for the block, primary, and secondary antibodies? What blocking agent and secondary antibody were used?

    I will be happy to assist you further once I have this additional information.

    Read More

    Question

    Dear Sir or Madam,
    I´d like to contact you for some questions about the primary antibodies offered by your company:
    Anti-Spastin antibody [Sp 3G11/1] (ab31850)
    Anti-Spastin antibody [Sp 6C6] (ab77144)
    Could you please tell me, which epitop on spastin is recognized by those primary antibodies? Or could you at least narrow down the location of recognition? Unfortunately, there are no informations about that.
    Thank you very much for your help and your reply.

    Read More
    Answer

    First of all I would like to apologise for the delay in getting back to you. I really appreciate your patience and comprehension.

    I contacted my lab colleagues and unfortunately there isn’t any information available about the epitope recognised by these antibodies, as they have not been mapped.

    Please let me know if there is anything else I can help you with.

    Read More

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