Overview

  • Product name
  • Description
    Goat polyclonal to SIAH1
  • Host species
    Goat
  • Tested applications
    Suitable for: IHC-P, WB, IP, ELISAmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Cow, Dog
  • Immunogen

    Synthetic peptide corresponding to Human SIAH1 aa 2-16 (N terminal).
    Sequence:

    SRQTATALPTGTSKC


    Database link: Q8IUQ4
    (Peptide available as ab22876)

  • Positive control
    • Human liver lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab2237 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
IHC-P 1/50. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. PubMed: 17189520

1:300 donkey anti-goat was used for secondary antibody.

WB Use a concentration of 1 - 3 µg/ml. Detects a band of approximately 28, 30 kDa (predicted molecular weight: 31, 35 kDa).Can be blocked with Human SIAH1 peptide (ab22876).
IP Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.

Target

  • Function
    E3 ubiquitin-protein ligase that mediates ubiquitination and subsequent proteasomal degradation of target proteins. E3 ubiquitin ligases accept ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates. Mediates E3 ubiquitin ligase activity either through direct binding to substrates or by functioning as the essential RING domain subunit of larger E3 complexes. Triggers the ubiquitin-mediated degradation of many substrates, including proteins involved in transcription regulation (MYB, POU2AF1, PML and RBBP8), a cell surface receptor (DCC), the cell-surface receptor-type tyrosine kinase FLT3, the cytoplasmic signal transduction molecules (KLF10/TIEG1 and NUMB), an antiapoptotic protein (BAG1), a microtubule motor protein (KIF22), a protein involved in synaptic vesicle function in neurons (SYP), a structural protein (CTNNB1) and SNCAIP. Confers constitutive instability to HIPK2 through proteasomal degradation. It is thereby involved in many cellular processes such as apoptosis, tumor suppression, cell cycle, axon guidance, transcription regulation, spermatogenesis and TNF-alpha signaling. Has some overlapping function with SIAH2. Induces apoptosis in cooperation with PEG3. Upon nitric oxid (NO) generation that follows apoptotic stimulation, interacts with S-nitrosylated GAPDH, mediating the translocation of GAPDH to the nucleus. GAPDH acts as a stabilizer of SIAH1, facilitating the degradation of nuclear proteins.
  • Tissue specificity
    Widely expressed at a low level. Down-regulated in advanced hepatocellular carcinomas.
  • Pathway
    Protein modification; protein ubiquitination.
  • Sequence similarities
    Belongs to the SINA (Seven in absentia) family.
    Contains 1 RING-type zinc finger.
    Contains 1 SIAH-type zinc finger.
  • Domain
    The RING-type zinc finger domain is essential for ubiquitin ligase activity.
    The SBD domain (substrate-binding domain) mediates the homodimerization and the interaction with substrate proteins. It is related to the TRAF family.
  • Post-translational
    modifications
    Phosphorylated on Ser-19 by ATM and ATR. This phosphorylation disrupts SIAH1 interaction with HIPK2, and subsequent proteasomal degradation of HIPK2.
  • Cellular localization
    Cytoplasm. Nucleus. Predominantly cytoplasmic. Partially nuclear.
  • Information by UniProt
  • Database links
  • Alternative names
    • E3 ubiquitin-protein ligase SIAH1 antibody
    • FLJ08065 antibody
    • hSIAH1 antibody
    • HUMSIAH antibody
    • Seven in absentia homolog 1 (Drosophila) antibody
    • Seven in absentia homolog 1 antibody
    • Siah 1 antibody
    • Siah 1a antibody
    • Siah E3 ubiquitin protein ligase 1 antibody
    • Siah-1 antibody
    • Siah-1a antibody
    • Siah1 antibody
    • SIAH1_HUMAN antibody
    • SIAH1A antibody
    • Ubiquitin ligase SIAH1 antibody
    see all

Images

  • All lanes : Anti-SIAH1 antibody (ab2237) at 1 µg/ml

    Lane 1 : Mouse brain lysate
    Lane 2 : Rat brain lysate
    Lane 3 : Mouse liver lysate
    Lane 4 : Rat liver lysate

    Lysates/proteins at 35 µg per lane.

    Predicted band size: 31, 35 kDa

  • Anti-SIAH1 antibody (ab2237) at 1 µg/ml + Human liver lysate at 35 µg

    Predicted band size: 31, 35 kDa

References

This product has been referenced in:
  • Wu W  et al. Tp53 Mutation Inhibits Ubiquitination and Degradation of WISP1 via Down-Regulation of Siah1 in Pancreatic Carcinogenesis. Front Pharmacol 9:857 (2018). Read more (PubMed: 30123132) »
  • Van der Hoek KH  et al. Viperin is an important host restriction factor in control of Zika virus infection. Sci Rep 7:4475 (2017). Read more (PubMed: 28667332) »
See all 16 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Question
Answer

Thank you for calling Abcam earlier today.

I am sorry about the issues that you have been having with ab2237 in western blot. As we discussed on the phone, I am sending you a vial of ab49177 as a free of charge replacement. Your new order number is * and you should receive the antibody tomorrow morning.

If there is anything else I can help you with, please let me know.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Jurkat cells)
Loading amount
10 µg
Specification
Jurkat cells
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C

Abcam user community

Verified customer

Submitted Oct 26 2011

Question

Our customers have problems of ab2237 antibody in western blot. The WB questionnarie is attached with mail. We look forward to hearing from you soon. • Antibody storage conditions (temperature/reconstitution etc) -20? 2. Please describe the problem (high background, wrong band size, more bands, no band etc). Right band size at 23 and 30 kDa with two unexpected bands. 3. On what material are you testing the antibody in WB? • Species: Human neuroblastoma cell line, SH-SY5Y. • Cell extract or Nuclear extract: Cell extract. • Purified protein or Recombinant protein: endogenous protein without purified. 3. The lysate • How much protein was loaded: unknown. • What lysis buffer was used: 2x SDS sample buffer. • What protease inhibitors were used: No. • What loading buffer was used: 2x SDS sample buffer. • Did you heat the samples: temperature and time: Heat at 95? 5 mins before loading. 4. Electrophoresis/Gel conditions/ Transfer conditions • Reducing or non reducing gel: Reducing gel. • Gel percentage : 12% • Transfer conditions: Towbin buffer. (24mM Tris-base, 192mM glycine, 20%methanol.) 5. Blocking conditions • Buffer: PBST • Blocking agent: milk, BSA, serum, what percentage: 5% • Incubation time: 30 mins • Incubation temperature: RT. 6. Primary Antibody • Specification (in which species was it raised against): Goat polyclonal to SIAH1 • At what dilution(s) have you tested this antibody: 1:1000 • What dilution buffer was used: PBST • Incubation time: 1 hr • Incubation temperature: RT • What washing steps were done: wash three times with PBST, each 10 mins. 7. Secondary Antibody • Specification (in which species was it raised against)? Rab poly to goat IgG. • At what dilution(s) have you tested this antibody: 1:10000 • Incubation time 1 hr • Wash steps: wash three times with PBST, each 10 mins. • Do you know whether the problems you are experiencing come from the secondary? No. 8. Detection method ECl, ECl+, other detection method: 9. Background bands • Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): No. • Is the blocking step sufficient? Yes, • Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) Yes. • At what size are the bands migrating? Could they be degradation products of your target? Unexpected bands at approximately 110kDa and 20kDa. • Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) 11. Did you apply positive and negative controls along with the samples? Please specify. No. 10. Optimization attempts • How many times have you tried the Western? Many times. • Do you obtain the same results every time e.g. are background bands always in the same place? No. • What steps have you altered? No.

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Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. This antibody is a relatively good selling antibody that we have received few complaints about. However, we do have one previous report of similar non-specific bands. I have read your customer's technical questionnaire and I have a few comments. This antibody is a polyclonal antibody that will show reactivity against proteins other than SIAH1 given long enough exposure under the appropriate conditions. Therefore at this stage like to make the following recommendations; I would like to suggest that your customer reduces the mass of protein they are using. This frequently results in more specific results. I would also like to recommend that your customer tries an overnight block using BSA as a blocking agent and replaces milk with 3% BSA in TBST during the antibody incubations. We frequently find that generates a significant improvement in the results that are obtained by western blotting when combined with a reduction in the mass of protein. Should this not lead to an improvement in the specificity of the results that your customer is obtaining please get back in touch with me.

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Answer

Thank you for your enquiry. To date we have not received any customer reviews relating to the use of this product so I am unsure exactly what the non specific bands are that you are seeing. Have you utilised protease inhibitors and eliminated the possibility that any lower band could be due to protein breakdown? Since the product is a pAb and will reorganise multiple epitopes it is also important to ensure that not too much protein was loaded onto the gel since proteins expressed in high levels will be non specifically detected by the pAb. I hope that this helps; unfortunately I am not able to give any more specific data relating to customer feedback.

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Question

. Order details: Product code: Anti-SIAH1 antibody AB2237 Batch number: 102802 Abcam order or Purchase order number: 543350 (Biozol) Antibody storage conditions (temperature/reconstitution etc): - 20?C 2. Please describe the problem (high background, wrong band size, more bands, no band etc). I could only detect the 45 kDa band and this is very light. Also I made a positive control like in the reference paper and the result was the same. 3. On what material are you testing the antibody in WB? Species: human cell line Cell extract or Nuclear extract: cell extract Purified protein or Recombinant protein: purified 3. The lysate How much protein was loaded: 30 ug What lysis buffer was used: Phosphosafe? Extraction Buffer (Novagen) or Sonification buffer (10 mM Tris/HCl, 1 mM EDTA, 1 mM mercaptoethanol, 5 % glycine) What protease inhibitors were used: aprotinine, leupeptine, PMSF What loading buffer was used: Laemmli-buffer Did you heat the samples: temperature and time: heat by 95 ?C for 5 minutes 4. Electrophoresis/Gel conditions/ Transfer conditions Reducing or non reducing gel: reducing gel Gel percentage : 10% SDS-polyacrylamide gel Transfer conditions: sandwich-method, 100 V, 60 minutes 5. Blocking conditions Buffer: PBS Blocking agent: milk, BSA, serum, what percentage: 5% non-fat dried milk Incubation time: over night Incubation temperature: 4 C 6. Primary Antibody Specification (in which species was it raised against): goat At what dilution(s) have you tested this antibody: 2 ?g/ml and 4 ?g/ml What dilution buffer was used: 5 % milk/ PBS/ 0,1 % Tween 20 Incubation time: 4 hours (room temperature) or over night (4 ?C) Incubation temperature: room temperature or 4 ?C What washing steps were done: 3 times washing for 10 minutes with PBS/ 0,1 % Tween, once again for 10 minutes with PBS 7. Secondary Antibody Specification (in which species was it raised against)? donkey (anti-goat IgG); IRDye700 conjugated At what dilution(s) have you tested this antibody: 1:10000 Incubation time: 1 hour Wash steps: the same like primary antibody Do you know whether the problems you are experiencing come from the secondary? No, because the secondary antibody worked correctly with all others primary antibodies I tested. 8. Detection method ECl, ECl+, other detection method: Odyssey Infrared Imaging System (LI-COR Biosciences) 9. Background bands Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a ?No primary? control): no background band of the secondary antibody Is the blocking step sufficient? yes Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) yes At what size are the bands migrating? Could they be degradation products of your target? It is the 45 kDa band like it is named in your datasheet, but no 30 kDa band. 10. Did you apply positive and negative controls along with the samples? Please specify. Positive control like in the reference paper: MCF-7 cell line induced with doxorubicin under the same conditions 11. Optimization attempts How many times have you tried the Western? 8 times Do you obtain the same results every time e.g. are background bands always in the same place? Yes, always the 45 kDa band only What steps have you altered? Longer incubation time, higher concentration of the antibody, made a positive control

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Answer

I'm sorry to hear you are having a problem with ab2237 not detecting both bands. The two bands were detected in human brain lysate (the strongest was at 30kDa and faint at 45kDa) and it may be that the customer's cells express more of the 45kDa form than the 30kDa. I could just see a faint band at 30kDa and maybe with the following tips your customer will get better signal for both bands: -we recommend using RIPA buffer for optimal extraction of the protein (with a complete Roche cocktail of inhibitors for maximal protease protection) -We used ECL+ to detect the signal, it is possible that another detection method needs higher concentrations of the primary (e.g 5ug/ml) and secondary -we recommend using TBST rather than PBST as this gives cleaner and sharper signals, to dilute antibodies and wash the membrane -the main problem could be too much blocking, which prevents adequate binding of the primary antibody. Please try 1hr blocking in 5% milk or 5%BSA, then incubate the primary overnight at 4C in TBST only (and also try in 0.1% milk) and the secondary in TBST only. Please be assured that if the customer runs our guaranteed positive control of human brain lysate and finds similar results with those modifications we will gladly offer a replacement or refund if the antibody was purchased in the last 90days,

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Answer

The originator of ab2237 has confirmed for me that the antibody was characterized using human brain lysate (NOT U937 whole cell lysate). If you have any additional questions, please contact us again.

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Answer

Thank you for your phone call. The datasheet for ab2287 is a bit confusing as it mentions both U937 lysate (U937 cells are a human cell line established from a diffuse histiocytic lymphoma and displaying many monocytic characteristics. It serves as an in vitro model for monocyte/macrophage differentiation) and human brain lysate. I have to contact the originator of this antibody for clarification and will get beck to you once I have hear back.

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Answer

Two bands are detected with this antibody. The 30k band is much stronger and the size agrees with published reports. We don't know what the 45k band is.

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Question
Answer

No, Abcam does not source any of its products from Santa Cruz. This is a totally unique antibody that has recently been generated. If you do purchase it, please let us know how you get on and of course Abcam guarantees the product will work as it says in the datasheet or a replacement or money back will be given..... no quibble!

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