Overview

  • Product name

    Anti-Spastin antibody [Sp 3G11/1]
    See all Spastin primary antibodies
  • Description

    Mouse monoclonal [Sp 3G11/1] to Spastin
  • Host species

    Mouse
  • Tested applications

    Suitable for: WB, ELISA, IHC-P, ICC/IFmore details
  • Species reactivity

    Reacts with: Rat, Human
  • Immunogen

    Recombinant full length protein (Human).

  • Positive control

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

Properties

Applications

Our Abpromise guarantee covers the use of ab31850 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 1/500. 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.
ELISA Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration. PubMed: 21757506
ICC/IF Use at an assay dependent concentration. PubMed: 21757506

Target

  • Relevance

    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.
  • Cellular localization

    Cytoplasm, Cytoskeleton, Endoplasmic reticulum, Endosome, Membrane, Microtubule, Nucleus
  • Database links

  • Alternative names

    • 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

Images

  • 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.
  • 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.

References

This product has been referenced in:

  • 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 . Read more (PubMed: 21757506) »
  • Salinas S  et al. Human spastin has multiple microtubule-related functions. J Neurochem 95:1411-20 (2005). Read more (PubMed: 16219033) »
See all 2 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

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

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

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

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

Sign up