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Anti-SIAH1抗体(ab2237)

Reviews (1)Q&A (8)References (23)
Western blot - Anti-SIAH1 antibody (ab2237)
  • Western blot - Anti-SIAH1 antibody (ab2237)
  • Immunocytochemistry/ Immunofluorescence - Anti-SIAH1 antibody (ab2237)
  • Immunocytochemistry/ Immunofluorescence - Anti-SIAH1 antibody (ab2237)

Key features and details

  • Goat polyclonal to SIAH1
  • Suitable for: WB, ICC/IF
  • Reacts with: Rat, Human
  • Isotype: IgG

概述

  • 产品名称

    Anti-SIAH1抗体
    参阅全部 SIAH1 一抗

  • 描述

    山羊多克隆抗体to SIAH1

  • 宿主

    Goat

  • 经测试应用

    适用于: WB, ICC/IFmore details

  • 种属反应性

    与反应: Rat, Human
    预测可用于: Mouse, Cow, Dog

  • 免疫原

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

    SRQTATALPTGTSKC


    Database link: Q8IUQ4
    (Peptide available as ab22876)
    Run BLAST with BLAST the sequence with ExPASy Run BLAST with BLAST the sequence with NCBI

  • 阳性对照

    • WB: Rat liver lysate, human liver lysate. ICC/IF- U2OS cells and HepG2 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ab2237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)
Use a concentration of 1 - 3 µg/ml. Predicted molecular weight: 31 kDa.

1 hour primary incubation at room temperature is recommended for this product.
ICC/IF
Use a concentration of 10 µg/ml.
说明
WB
Use a concentration of 1 - 3 µg/ml. Predicted molecular weight: 31 kDa.

1 hour primary incubation at room temperature is recommended for this product.
ICC/IF
Use a concentration of 10 µg/ml.

靶标

  • 功能

    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.

  • 组织特异性

    Widely expressed at a low level. Down-regulated in advanced hepatocellular carcinomas.

  • 通路

    Protein modification; protein ubiquitination.

  • 序列相似性

    Belongs to the SINA (Seven in absentia) family.
    Contains 1 RING-type zinc finger.
    Contains 1 SIAH-type zinc finger.

  • 结构域

    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.

  • 翻译后修饰

    Phosphorylated on Ser-19 by ATM and ATR. This phosphorylation disrupts SIAH1 interaction with HIPK2, and subsequent proteasomal degradation of HIPK2.

  • 细胞定位

    Cytoplasm. Nucleus. Predominantly cytoplasmic. Partially nuclear.
  • Information by UniProt

  • 数据库链接

    see all

  • 别名

    • 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

图片

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

    Predicted band size: 31 kDa
    Observed band size: 37 kDa why is the actual band size different from the predicted?



    Primary incubation 1 hour at room temperature. Detected by chemiluminescence. RIPA buffer was used.

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

    Predicted band size: 31 kDa
    Observed band size: 30, 37 kDa why is the actual band size different from the predicted?



    Primary incubation 1 hour at room temperature. Detected by chemiluminescence. RIPA buffer was used.

  • Immunocytochemistry/ Immunofluorescence - Anti-SIAH1 antibody (ab2237)
    Immunocytochemistry/ Immunofluorescence - Anti-SIAH1 antibody (ab2237)

    Immunofluorescence analysis of paraformaldehyde fixed, 0.15% Triton permeabilized HepG2 (human liver hepatocellular carcinoma cell) cells labelling SIAH1 with primary anti-SIAH1 antibody (ab2237) at 10ug/ml for 1 hour, followed by Alexa Fluor 488 secondary antibody at 2ug/ml. Image showing nuclear and cytoplasmic staining. The nuclear counterstain is DAPI (blue).

    Negative control: Unimmunized goat IgG at 10ug/ml followed by Alexa Fluor 488 secondary antibody.

  • Immunocytochemistry/ Immunofluorescence - Anti-SIAH1 antibody (ab2237)
    Immunocytochemistry/ Immunofluorescence - Anti-SIAH1 antibody (ab2237)

    Immunofluorescence analysis of paraformaldehyde fixed, 0.15% Triton permeabilized U2OS (human bone osteosarcoma epithelial cell) cells labelling SIAH1 with primary anti-SIAH1 antibody (ab2237) at 10ug/ml for 1 hour, followed by Alexa Fluor 488 secondary antibody at 2ug/ml. Image showing nuclear and cytoplasmic staining. The nuclear counterstain is DAPI (blue).

    Negative control: Unimmunized goat IgG at 10ug/ml followed by Alexa Fluor 488 secondary antibody.

文献 (23)

发表研究结果有使用 ab2237？请让我们知道，以便我们可以引用本数据表中的参考文章。

ab2237 被引用在 23 文献中.

  • Ayo-Martin AC  et al. GNG5 Controls the Number of Apical and Basal Progenitors and Alters Neuronal Migration During Cortical Development. Front Mol Biosci 7:578137 (2020). PubMed: 33330619
  • Shani V  et al. Physiological and pathological roles of LRRK2 in the nuclear envelope integrity. Hum Mol Genet 28:3982-3996 (2019). PubMed: 31626293
  • Shrestha R  et al. Aberrant hiPSCs-Derived from Human Keratinocytes Differentiates into 3D Retinal Organoids that Acquire Mature Photoreceptors. Cells 8:N/A (2019). PubMed: 30634512
  • 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). PubMed: 30123132
  • Deng Q  et al. PHF19 promotes the proliferation, migration, and chemosensitivity of glioblastoma to doxorubicin through modulation of the SIAH1/ß-catenin axis. Cell Death Dis 9:1049 (2018). PubMed: 30323224
View all Publications for this product

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1-9 of 9 Abreviews or Q&A

Western blot abreview for Anti-SIAH1 antibody

 Excellent
Abreviews
abreview image
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
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The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

提交于 Oct 26 2011

Question

No signal in WB on rat heart samples.

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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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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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Question

We have tried your Siah1 antibody on Neuro2a cell lysates. Although it recognizes two bands corresponding to the Siah1a and 1b, but it also shows at least two other bands at ~20 and 110 kDa. I wonder if anyone has seen these two bands before.

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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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Question

Datasheet says: Western Blot Approx 30kDa and 45kDa bands seen in U937 Human Brain lysate. In the caption for the image, it states that Human Brain lysate was used. Was U937 whole cell lysate used to characterize this antibody, or human brain lysate? Please confirm.

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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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Question

Datasheet says: Western Blot Approx 30kDa and 45kDa bands seen in U937 Human Brain lysate. In the caption for the image, it states that Human Brain lysate was used. Was U937 whole cell lysate used to characterize this antibody, or human brain lysate? Please confirm.

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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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Question

Customer is using ab2237 in Western blotting and would like to know the following concerning the image on the datasheet - it states that approx 30kDa and 45kDa bands are seen, but there appears to be 3 bands, any more information regarding this? Of the 3, which band is SIAH1?

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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

Is this antibody made by Santa Cruz?

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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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