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Anti-pan Arrestin 抗体 (ab2914)

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Reviews (1)Q&A (5)References (15)

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Western blot - Anti-pan Arrestin antibody (ab2914)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan Arrestin antibody (ab2914)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan Arrestin antibody (ab2914)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan Arrestin antibody (ab2914)
  • Western blot - Anti-pan Arrestin antibody (ab2914)

Key features and details

  • Rabbit polyclonal to pan Arrestin
  • Suitable for: WB, IHC-P
  • Reacts with: Rat, Human
  • Isotype: IgG

こちらの製品もご検討ください

二次抗体
Product image
Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
ノックアウト
Human ARRB1 knockout A549 cell line (ab277839)

関連製品

製品の概要

  • 製品名

    Anti-pan Arrestin antibody
  • 製品の詳細

    Rabbit polyclonal to pan Arrestin
  • 由来種

    Rabbit
  • 特異性

    Ab2914 detects recombinant rat beta-arrestin and beta-arrestin2 (not tested against endogenous protein). This antibody does not detect visual or cone arrestin.
  • アプリケーション

    適用あり: WB, IHC-Pmore details
  • 種交差性

    交差種: Rat, Human
    交差が予測される動物種: Mouse, Rabbit, Cynomolgus monkey, Rainbow trout
  • 免疫原

    Synthetic peptide corresponding to Human pan Arrestin aa 384-397.
    Sequence:

    DDIVFEDFARLRLK


    (Peptide available as ab4932)
    Run BLAST with BLAST the sequence with ExPASy Run BLAST with BLAST the sequence with NCBI
  • 特記事項

    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

製品の特性

  • 製品の状態

    Liquid
  • 保存方法

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • バッファー

    Preservative: 0.05% Sodium azide
    Constituents: 0.1% BSA, 99% PBS
  • Concentration information loading...
  • 精製度

    Immunogen affinity purified
  • 一次抗体 備考

    Vision involves the conversion of light into electrochemical signals that are processed by the retina and subsequently sent to and interpreted by the brain. The process of converting light to an electrochemical signal begins when the membrane-bound protein, rhodopsin, absorbs light within the retina. Photoexcitation of rhodopsin causes the cytoplasmic surface of the protein to become catalytically active. In the active state, rhodopsin activates transducin, a GTP binding protein. Once activated, transducin promotes the hydrolysis of cGMP by phosphodiesterase (PDE). The decrease of intracellular cGMP concentrations causes the ion channels within the outer segment of the rod or cone to close, thus causing membrane hyperpolarization and, eventually, signal transmission. Rhodopsin’s activity is believed to be shut off by its phosphorylation followed by binding of the soluble protein arrestin. Arrestins are cytosolic proteins that are involved in G protein-coupled receptor (GPCR) desensitization. Arrestin binding to activated GPCRs is phosphorylation dependent and, once bound, uncouple the GPCR from the associated heterotrimeric G proteins. There are currently 4 known mammalian isoforms, beta-arrestin1 (arrestin2), beta-arrestin2 (arrestin3), visual arrestin (arrestin1), and cone arrestin. The beta- isoforms are ubiquitously expressed and are known to interact with acetylcholine and adrenergic receptors. Visual and cone arrestins are found to interact directly with transducin.
  • ポリ/モノ

    ポリクローナル
  • アイソタイプ

    IgG
  • 研究分野

    • Neuroscience
    • Neurotransmission
    • Receptors / Channels
    • GPCR
    • More GPCR
    • Signal Transduction
    • Signaling Pathway
    • G Protein Signaling
    • Heterotrimeric G Proteins
    • Regulators
    • Neuroscience
    • Sensory System
    • Visual system

関連製品

  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
  • Immunizing Peptide (Blocking)

    • Human pan Arrestin peptide (ab4932)
  • Isotype control

    • Rabbit IgG, polyclonal - Isotype Control (ChIP Grade) (ab171870)

アプリケーション

The Abpromise guarantee

Abpromise保証は、 次のテスト済みアプリケーションにおけるab2914の使用に適用されます

アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。

アプリケーション Abreviews 特記事項
WB
Use a concentration of 3 µg/ml. Detects a band of approximately 47, 49 kDa.Can be blocked with Human pan Arrestin peptide (ab4932).
IHC-P
1/100 - 1/1000.
特記事項
WB
Use a concentration of 3 µg/ml. Detects a band of approximately 47, 49 kDa.Can be blocked with Human pan Arrestin peptide (ab4932).
IHC-P
1/100 - 1/1000.

ターゲット情報

  • 機能

    Functions in regulating agonist-mediated G-protein coupled receptor (GPCR) signaling by mediating both receptor desensitization and resensitization processes. During homologous desensitization, beta-arrestins bind to the GPRK-phosphorylated receptor and sterically preclude its coupling to the cognate G-protein; the binding appears to require additional receptor determinants exposed only in the active receptor conformation. The beta-arrestins target many receptors for internalization by acting as endocytic adapters (CLASPs, clathrin-associated sorting proteins) and recruiting the GPRCs to the adapter protein 2 complex 2 (AP-2) in clathrin-coated pits (CCPs). However, the extent of beta-arrestin involvement appears to vary significantly depending on the receptor, agonist and cell type. Internalized arrestin-receptor complexes traffic to intracellular endosomes, where they remain uncoupled from G-proteins. Two different modes of arrestin-mediated internalization occur. Class A receptors, like ADRB2, OPRM1, ENDRA, D1AR and ADRA1B dissociate from beta-arrestin at or near the plasma membrane and undergo rapid recycling. Class B receptors, like AVPR2, AGTR1, NTSR1, TRHR and TACR1 internalize as a complex with arrestin and traffic with it to endosomal vesicles, presumably as desensitized receptors, for extended periods of time. Receptor resensitization then requires that receptor-bound arrestin is removed so that the receptor can be dephosphorylated and returned to the plasma membrane. Involved in internalization of P2RY4 and UTP-stimulated internalization of P2RY2. Involved in phosphorylation-dependent internalization of OPRD1 ands subsequent recycling. Involved in the degradation of cAMP by recruiting cAMP phosphodiesterases to ligand-activated receptors. Beta-arrestins function as multivalent adapter proteins that can switch the GPCR from a G-protein signaling mode that transmits short-lived signals from the plasma membrane via small molecule second messengers and ion channels to a beta-arrestin signaling mode that transmits a distinct set of signals that are initiated as the receptor internalizes and transits the intracellular compartment. Acts as signaling scaffold for MAPK pathways such as MAPK1/3 (ERK1/2). ERK1/2 activated by the beta-arrestin scaffold is largely excluded from the nucleus and confined to cytoplasmic locations such as endocytic vesicles, also called beta-arrestin signalosomes. Recruits c-Src/SRC to ADRB2 resulting in ERK activation. GPCRs for which the beta-arrestin-mediated signaling relies on both ARRB1 and ARRB2 (codependent regulation) include ADRB2, F2RL1 and PTH1R. For some GPCRs the beta-arrestin-mediated signaling relies on either ARRB1 or ARRB2 and is inhibited by the other respective beta-arrestin form (reciprocal regulation). Inhibits ERK1/2 signaling in AGTR1- and AVPR2-mediated activation (reciprocal regulation). Is required for SP-stimulated endocytosis of NK1R and recruits c-Src/SRC to internalized NK1R resulting in ERK1/2 activation, which is required for the antiapoptotic effects of SP. Is involved in proteinase-activated F2RL1-mediated ERK activity. Acts as signaling scaffold for the AKT1 pathway. Is involved in alpha-thrombin-stimulated AKT1 signaling. Is involved in IGF1-stimulated AKT1 signaling leading to increased protection from apoptosis. Involved in activation of the p38 MAPK signaling pathway and in actin bundle formation. Involved in F2RL1-mediated cytoskeletal rearrangement and chemotaxis. Involved in AGTR1-mediated stress fiber formation by acting together with GNAQ to activate RHOA. Appears to function as signaling scaffold involved in regulation of MIP-1-beta-stimulated CCR5-dependent chemotaxis. Involved in attenuation of NF-kappa-B-dependent transcription in response to GPCR or cytokine stimulation by interacting with and stabilizing CHUK. May serve as nuclear messenger for GPCRs. Involved in OPRD1-stimulated transcriptional regulation by translocating to CDKN1B and FOS promoter regions and recruiting EP300 resulting in acetylation of histone H4. Involved in regulation of LEF1 transcriptional activity via interaction with DVL1 and/or DVL2 Also involved in regulation of receptors other than GPCRs. Involved in Toll-like receptor and IL-1 receptor signaling through the interaction with TRAF6 which prevents TRAF6 autoubiquitination and oligomerization required for activation of NF-kappa-B and JUN. Binds phosphoinositides. Binds inositolhexakisphosphate (InsP6) (By similarity). Involved in IL8-mediated granule release in neutrophils.
  • 配列類似性

    Belongs to the arrestin family.
  • ドメイン

    The [DE]-X(1,2)-F-X-X-[FL]-X-X-X-R motif mediates interaction the AP-2 complex subunit AP2B1 (By similarity). Binding to phosphorylated GPCRs induces a conformationanl change that exposes the motif to the surface.
    The N-terminus binds InsP6 with low affinity.
    The C-terminus binds InsP6 with high affinity.
  • 翻訳後修飾

    Constitutively phosphorylated at Ser-412 in the cytoplasm. At the plasma membrane, is rapidly dephosphorylated, a process that is required for clathrin binding and ADRB2 endocytosis but not for ADRB2 binding and desensitization. Once internalized, is rephosphorylated.
    The ubiquitination status appears to regulate the formation and trafficking of beta-arrestin-GPCR complexes and signaling. Ubiquitination appears to occur GPCR-specific. Ubiquitinated by MDM2; the ubiquitination is required for rapid internalization of ADRB2. Deubiquitinated by USP33; the deubiquitination leads to a dissociation of the beta-arrestin-GPCR complex. Stimulation of a class A GPCR, such as ADRB2, induces transient ubiquitination and subsequently promotes association with USP33.
  • 細胞内局在

    Cytoplasm. Nucleus. Cell membrane. Membrane > clathrin-coated pit. Cell projection > pseudopodium. Cytoplasmic vesicle. Translocates to the plasma membrane and colocalizes with antagonist-stimulated GPCRs. The monomeric form is predominantly located in the nucleus. The oligomeric form is located in the cytoplasm. Translocates to the nucleus upon stimulation of OPRD1.
  • Target information above from: UniProt accession P49407 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • 参照データベース

    • Entrez Gene: 408 Human
    • Entrez Gene: 409 Human
    • Entrez Gene: 109689 Mouse
    • Entrez Gene: 25387 Rat
    • Omim: 107940 Human
    • Omim: 107941 Human
    • SwissProt: P32121 Human
    • SwissProt: P49407 Human
    • SwissProt: Q8BWG8 Mouse
    • SwissProt: P29066 Rat
    • Unigene: 503284 Human
    • Unigene: 625320 Human
    • Unigene: 260193 Mouse
    • Unigene: 34876 Rat
    see all
  • 別名

    • ARB1 antibody
    • ARB2 antibody
    • ARR1 antibody
    • ARR2 antibody
    • ARRB1 antibody
    • ARRB1_HUMAN antibody
    • Arrestin beta 1 antibody
    • Arrestin beta 2 antibody
    • Arrestin beta-1 antibody
    • Beta-arrestin-1 antibody
    see all

画像

  • Western blot - Anti-pan Arrestin antibody (ab2914)
    Western blot - Anti-pan Arrestin antibody (ab2914)
    All lanes : Anti-pan Arrestin antibody (ab2914) at 1/1000 dilution

    Lane 1 : C6 cell lysate
    Lane 2 : Rat brain cell lysate
    Lane 3 : HeLa cell lysate

    Lysates/proteins at 25 µg per lane.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan Arrestin antibody (ab2914)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan Arrestin antibody (ab2914)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of pan Arrestin showing staining in the cytoplasm and nucleus of paraffin-treated human cerebellum tissue (right) compared with a negative control in the absence of primary antibody (left). Antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then incubated with ab2914 diluted by 3% BSA-PBS at a dilution of 1:500 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan Arrestin antibody (ab2914)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan Arrestin antibody (ab2914)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of pan Arrestin showing staining in the cytoplasm and nucleus of paraffin-treated rat brain tissue (right) compared with a negative control in the absence of primary antibody (left). Antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then incubated with ab2914 diluted by 3% BSA-PBS at a dilution of 1:500 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan Arrestin antibody (ab2914)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan Arrestin antibody (ab2914)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of pan Arrestin showing staining in the cytoplasm and nucleus of paraffin-treated rat spleen tissue (right) compared with a negative control in the absence of primary antibody (left). Antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then incubated with ab2914 diluted by 3% BSA-PBS at a dilution of 1:500 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

  • Western blot - Anti-pan Arrestin antibody (ab2914)
    Western blot - Anti-pan Arrestin antibody (ab2914)
    Anti-pan Arrestin antibody (ab2914) at 1 µg/ml + Human brain tissue lysate - total protein (ab29466) at 10 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Observed band size: 49 +51 kDa why is the actual band size different from the predicted?
    Additional bands at: 27 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 90 seconds


    pan Arrestin contains a number of potential phosphorylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

プロトコール

  • Immunohistochemistry protocols
  • Immunocytochemistry & immunofluorescence protocols
  • Western blot protocols

Click here to view the general protocols

データシートおよび資料

  • SDS download

  • Datasheet download

    Download

参考文献 (15)

ab2914 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

ab2914 は 15 報の論文で使用されています。

  • Hutchinson JL  et al. Arrestin-3 differentially regulates platelet GPCR subsets. Platelets 31:641-645 (2020). PubMed: 31684789
  • Bailes HJ  et al. Optogenetic interrogation reveals separable G-protein-dependent and -independent signalling linking G-protein-coupled receptors to the circadian oscillator. BMC Biol 15:40 (2017). PubMed: 28506231
  • Dela Paz NG  et al. Shear stress induces Gaq/11 activation independently of G protein-coupled receptor activation in endothelial cells. Am J Physiol Cell Physiol 312:C428-C437 (2017). PubMed: 28148497
  • Luessen DJ  et al. RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells. Neuropharmacology 110:297-307 (2016). PubMed: 27528587
  • Green JA  et al. Recruitment of ß-Arrestin into Neuronal Cilia Modulates Somatostatin Receptor Subtype 3 Ciliary Localization. Mol Cell Biol 36:223-35 (2016). PubMed: 26503786
View all Publications for this product

レビューと Q&A

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レビューを送る 質問を送る

1-6 of 6 Abreviews or Q&A

Question

Inquiry: i want to know soon details about anti-arrestin and who distribuite your kits in italy.thanks

Read More

Abcam community

Verified customer

Asked on May 23 2012

Answer

Thank you for your enquiry.

We have 3 antibodies that may be of interest:

ab2914 Anti-pan Arrestin antibody
www.abcam.com/ab2914

ab15852 Anti-Arrestin C antibody
www.abcam.com/ab15852

ab69599 Anti-Arrestin C antibody
www.abcam.com/ab69599

We have 2 distributors in Italy:

Prodotti Gianni www.prodottigianni.com

Aurogene www.aurogene.com

Please dont hesitate to contact me should you have further questions.

Read More

Abcam Scientific Support

Answered on May 23 2012

Immunocytochemistry/ Immunofluorescence abreview for Anti-pan Arrestin antibody

Good
Abreviews
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HEK 293)
Specification
HEK 293
Fixative
Paraformaldehyde
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3%
Read More

Mrs. Danijela Markovic

Verified customer

投稿 Aug 30 2006

Question

BATCH NUMBER 118458 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM wrong band size - expected 47/49 kDa, but only two strong bands at ~83 and 40 kDa, and a few fainter bands, but none of the expected size. SAMPLE cell lysates - SK-N-MC, H295R PRIMARY ANTIBODY primary antibody used 1:333 in milk in TBS tween, incubated overnight at 4 degrees C with agitation. blots were washed 3 times at room temperature with agitation for 10-20 min per wash. DETECTION METHOD ECLPlus ANTIBODY STORAGE CONDITIONS reconstituted in water, aliquots stored in -20 degrees C SAMPLE PREPARATION protease inhibitor added, samples boiled for 5 min in SDS lysis buffer AMOUNT OF PROTEIN LOADED 50 ug ELECTROPHORESIS/GEL CONDITIONS 10% reducing gel TRANSFER AND BLOCKING CONDITIONS blocked with milk in TBS tween for 1 hour at room temperature, with agitation SECONDARY ANTIBODY 1:5000 in milk in TBS tween, incubated for 1 hour at room temperature. blots were washed 3 times at room temperature with agitation for 10-20 min per wash. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

Read More

Abcam community

Verified customer

Asked on Nov 08 2005

Answer

Thank you very much for your protocol details, I think there may be a step which will also help you (as well as running the positive controls mentioned in my previous e-mail-brain and spleen). In our experience multiple non specific bands can be caused by too much blocking as the milk causes the antibody to bind to lots of proteins. I would therefore recommend trying: -a different blocking agent: I have very good experience of 5%BSA in TBST (1hr, RT) and incubating the antibody in TBST only -less milk: try 30min incubation in 5%milk then rinse gently in TBST and incubate the antibody in TBST only at 4C. From the papers I have read about arrestin the protein is found in the cytoplasm so you should not need a very strong detergent to extract it, however I would recommend checking that the lysis buffer is fresh and the protease inhibitors too and samples are kept on ice at all times. Finally can I make sure the loading buffer contains reducing and denaturing agents? I hope the above advice will help you, please do not hesitate to contact me again if you need further assistance,

Read More

Abcam Scientific Support

Answered on Nov 09 2005

Question

We have the rabbit polyclonal pan arrestin antibody (AB2914-200) of lot number 118458 and have used it for Western blotting. There are two strong bands on the blot, one of ~83 kDa and another of ~40 kDa, and a few faint bands. However there is nothing of the expected 47/49 kDa size. The lysates on the blots were of two human cell lines that do express beta-arrestin. Have you had feedback from other customers on this batch of the antibody? Regards,

Read More

Abcam community

Verified customer

Asked on Nov 04 2005

Answer

I have been informed this morning that the antibody was tested in WB against COS7 cell lysates overexpressing rat beta arrestin 2 (flag tagged) and have not therefore been tested against the endogenous protein. A literature search revealed that in human, beta arrestin 2 is primarily expressed in brain and spleen so these may be good positive controls to try. I hope this information will already help you and would like to offer more protocol advice if you can provide more protocol details. Maybe by increasing the amount of cell lysate loaded, increasing the amount of primary antibody, and/or increasing the incubation time and changing the blocking buffer you will be able to get a good specific band. I look forward to receiving your protocol details to be able to better help you,

Read More

Abcam Scientific Support

Answered on Nov 08 2005

Question

We have the rabbit polyclonal pan arrestin antibody (AB2914-200) of lot number 118458 and have used it for Western blotting. There are two strong bands on the blot, one of ~83 kDa and another of ~40 kDa, and a few faint bands. However there is nothing of the expected 47/49 kDa size. The lysates on the blots were of two human cell lines that do express beta-arrestin. Have you had feedback from other customers on this batch of the antibody? Regards,

Read More

Abcam community

Verified customer

Asked on Nov 04 2005

Answer

I'm sorry to hear you are experiencing problem with ab2914. We have not received other complaints since 2004 about this antibody and have not tested it in human samples but having done a BLAST search I found 100% homology of the immunogen with the human protein so I would expect it to work . I would like to recommend to run a positive control along your samples and I am in touch with the source of the antibody to find out what they recommend, my apologies for the delay. Without a detailed protocol it is difficult to know that the problem might be so if you please click on the link below this will take you to a protocol questionnaire to help you put your details together and add "FAO Sarah" in the additional notes I will look at your protocol as soon as I can, https://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=2914&mode=questionaire I look forward to hearing from you to help you more,

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Abcam Scientific Support

Answered on Nov 07 2005

Question

ANTIBODY CODE ab2914 BATCH NUMBER 28002 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal at the 45-55 kDa range- no bands whatsover in this range. Non specific band at 70 and 30 kDa. MW markers known to be good. SAMPLE We have tested 3 separate blots. The first was with Mouse brain tissue homogenates in RIPA (NP40, Deoxycholate). The second was with mouse brain tissue homogenates in RIPA (SDS). The third was from an IP from mouse brain previously blotted for Barrestin1 (in house antibody, very selective for Barr1- band was there- Abcam antibody did not detect it). On one of the blots, lysates from HEK-293 cells were also included. Barr1 AB detects this human form- the Abcam AB did not detect it again. PRIMARY ANTIBODY We have tested 1:500, 1:250 and 1:100 dilutions of the AB- SECONDARY ANTIBODY The secondary AB has been used with the other Barr1 ab successfully. We use a 1:2000 dilution, 1 hour- anti Rabbit Jackson immunoresearch labs DETECTION METHOD ecl- Pierce- This all works for the other Barrestin AB- POSITIVE AND NEGATIVE CONTROLS USED The other Barr1 AB detects the ~48 KDa band- nothing is present with the Abcam antibody on the same blots or on parallel blots. We really want to detect Barr2 as well- we hoped the Abcam AB would be useful for looking at both proteins simultaneously. However, we cannot detect either. ANTIBODY STORAGE CONDITIONS 4C- just received last week SAMPLE PREPARATION Brain regions or Cell lysates.- the real control is that we are able to detect Barr1 using another antibody on these same blots. It's not our tissue, blotting conditions, etc. AMOUNT OF PROTEIN LOADED We have tested 10 ug to 100 ug protein loaded. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? protein levels, dilutions, incubation times (o/n 4 degrees C at 1:100- still nothing.) ADDITIONAL NOTES The Barrestin ABs are notoriously nonspecific. we were hoping to be able to determine Barr1 vs. Barr2 via MW (which is possible). However- this antibody recognizes neither form in mouse or in human. It is odd considering the epitope is preserved in these species. Further, a comment on your website suggested it was good for human- Could we have a bad batch? Any assistance would be invaluable. Having a good Barrestin antibody would benefit more than ourselves.

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Abcam community

Verified customer

Asked on Mar 09 2004

Answer

I am sorry to hear that you are experiencing trouble with this antibody. I have enquired with the originator of ab2914 and everything appears fine with your protocol. We have had no other complaints about this antibody, but this particular antibody has not yet been tested with mouse so maybe it doesn't work in mouse.

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Abcam Scientific Support

Answered on Mar 11 2004

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