製品の概要

  • 製品名
    Anti-CaMKII alpha (phospho T286) antibody
    CaMKII alpha 一次抗体 製品一覧
  • 製品の詳細
    Rabbit polyclonal to CaMKII alpha (phospho T286)
  • 由来種
    Rabbit
  • 特異性
    It does cross-react with CAMKII beta protein
  • アプリケーション
    適用あり: IHC-Fr, ICC/IF, WB, IHC (PFA fixed)more details
  • 種交差性
    交差種: Mouse, Rat, Rabbit, Human
  • 免疫原

    Synthetic phosphopeptide derived from a region of human CaM Kinase II alpha that contains threonine 286.

  • ポジティブ・コントロール
    • Rat brain lysate.
  • 特記事項


    Calcium/calmodulin-dependent protein kinase II alpha (CaM Kinase II alpha) is a 50 kDa member of CaM Kinase II family of serine-threonine kinases that transduce Ca2+ signals to several target proteins, including ion channels and transcription activators. CaM Kinase II is predominantly expressed in two isoforms in the brain: alpha and beta. CaM Kinase II plays an important role in neuronal plasticity and memory formation, and exerts both calcium-calmodulin-dependent and -independent activities. Autophosphorylation of CaM Kinase II alpha on threonine 286 allows the kinase to switch from a calmodulin-dependent to a calmodulin-independent state, and is required for various cellular functions including hippocampal long-term potentiation (LTP), special learning, and hippocampus-dependent memory.

製品の特性

  • 製品の状態
    Liquid
  • 保存方法
    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • バッファー
    pH: 7.30
    Preservative: 0.05% Sodium azide
    Constituents: PBS, 50% Glycerol, 0.1% BSA
  • Concentration information loading...
  • 精製度
    Immunogen affinity purified
  • 特記事項(精製)
    The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated CaM Kinase II alpha. The final product is generated by affinity chromatography using a CaM Kinase II alpha-derived peptide that is phosphorylated at threonine 286.
  • 一次抗体 備考
    Calcium/calmodulin-dependent protein kinase II alpha (CaM Kinase II alpha) is a 50 kDa member of CaM Kinase II family of serine-threonine kinases that transduce Ca2+ signals to several target proteins, including ion channels and transcription activators. CaM Kinase II is predominantly expressed in two isoforms in the brain: alpha and beta. CaM Kinase II plays an important role in neuronal plasticity and memory formation, and exerts both calcium-calmodulin-dependent and -independent activities. Autophosphorylation of CaM Kinase II alpha on threonine 286 allows the kinase to switch from a calmodulin-dependent to a calmodulin-independent state, and is required for various cellular functions including hippocampal long-term potentiation (LTP), special learning, and hippocampus-dependent memory.
  • ポリ/モノ
    ポリクローナル
  • アイソタイプ
    IgG
  • 研究分野

アプリケーション

Our Abpromise guarantee covers the use of ab5683 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
IHC-Fr Use at an assay dependent concentration.
ICC/IF Use a concentration of 5 µg/ml.
WB 1/1000. Detects a band of approximately 50 kDa.
IHC (PFA fixed) 1/300.

ターゲット情報

  • 機能
    CaM-kinase II (CAMK2) is a prominent kinase in the central nervous system that may function in long-term potentiation and neurotransmitter release. Member of the NMDAR signaling complex in excitatory synapses it may regulate NMDAR-dependent potentiation of the AMPAR and synaptic plasticity.
  • 配列類似性
    Belongs to the protein kinase superfamily. CAMK Ser/Thr protein kinase family. CaMK subfamily.
    Contains 1 protein kinase domain.
  • 細胞内局在
    Cell junction > synapse > presynaptic cell membrane. Cell junction > synapse. Postsynaptic lipid rafts.
  • Information by UniProt
  • 参照データベース
  • 別名
    • Alpha CaMKII antibody
    • Calcium calmodulin dependent protein kinase II antibody
    • Calcium/calmodulin dependent protein kinase II alpha B subunit antibody
    • Calcium/calmodulin dependent protein kinase type II alpha chain antibody
    • Calcium/calmodulin-dependent protein kinase (CaM kinase) II alpha antibody
    • Calcium/calmodulin-dependent protein kinase II alpha antibody
    • Calcium/calmodulin-dependent protein kinase II-alpha antibody
    • Calcium/calmodulin-dependent protein kinase type II subunit alpha antibody
    • Calcium/calmodulin-dependent protein kinase type IIA antibody
    • CaM kinase II alpha chain antibody
    • CaM kinase II alpha subunit antibody
    • CaM kinase II subunit alpha antibody
    • CaMK II alpha subunit antibody
    • CaMK-II subunit alpha antibody
    • Camk2a antibody
    • CAMKA antibody
    • CaMKII antibody
    • CaMKIINalpha antibody
    • EC 2.7.11.17 antibody
    • KCC2A_HUMAN antibody
    • KIAA0968 antibody
    • MGC123320 antibody
    • MGC139375 antibody
    • MGC155201 antibody
    • mKIAA0968 antibody
    • PK2CDD antibody
    • PKCCD antibody
    • R74975 antibody
    • zgc:112538 antibody
    • zgc:123320 antibody
    see all

画像

  • Immunofluorescent staining for CaMKII alpha phospho (T286) using ab5683 (1/300, incubated for 18 hours) in rat spinal cord. To induce CaMKII alpha phospho (T286) protein expression, a noxious stimulus was administered to the rat 5 minutes prior to 4% PFA perfusion fixation (this is a known paradigm for inducing phosphorylation CaMKII in some spinal neurons). The resulting immunofluorescent staining for CaMKII alpha phospho (T286) protein is observed in the cytoplasm of many dorsal horn spinal neurons (surrounding the central canal [A] or in the ventral horn [B]). Omition of primary antibody resulted in a lack of staining (data not shown).
     
    Protocol details: Tissue was prepared by 4% paraformaldehyde cardiac perfusion fixation. Tissue was frozen on dry ice and then embedded in OCT compound and cut on cryostat. An antigen retrieval step was not neccesary for the IHC protocol.

  • ICC/IF image of ab5683 stained PC12 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab5683, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Peptide Competition and Phosphatase Treatment: Rat brain lysates were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-4) or treated with lambda (ë) phosphatase (5) and blocked with a 5% BSA-TBST buffer for one hour at room temperature, then incubated with ab5683 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine-containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal method. The data show that only the peptide corresponding to CaM Kinase II alpha [pT286] blocks the antibody signal. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific. Peptide Competiti
  • Primary Antibody 1/1000 24 hours at 4°C
    Secondary Antibody (1/5000): HRP-conjugated Goat polyclonal to rabbit IgG
    Blocking step: 5% BSA for 12 hours at 4°C

    See Abreview

参考文献

This product has been referenced in:
  • Zhao L  et al. Effects of intrathecal bupivacaine on the NR2B/CaMKIIa/CREB signaling pathway in the rat lumbar spinal cord. Mol Med Rep 17:4508-4514 (2018). Read more (PubMed: 29344649) »
  • Rhee J  et al. Impaired Hippocampal Synaptic Plasticity and Enhanced Excitatory Transmission in a Novel Animal Model of Autism Spectrum Disorders with Telomerase Reverse Transcriptase Overexpression. Mol Cells 41:486-494 (2018). Read more (PubMed: 29696935) »
See all 23 Publications for this product

レビューと Q&A

1-10 of 10 Abreviews or Q&A

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (10% separating gel)
Sample
Mouse Tissue lysate - whole (Hippocampal homogenate)
Specification
Hippocampal homogenate
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

投稿 Feb 13 2014

Answer

Thank you for your email.

For ab5683: I have checked the homology between the human CaMK2 alpha and rabbit CaMK2 delta around the phosphorylation site T286: It is 90-100% with maximal one residue mismatching. Thus the homology is very high, and the antibody should crossreact with the rabbit delta form. In any case, we would guarantee the antibody to work with rabbit samples.

Unfortunately, we do not carry an antibody to oxidized CaMK2.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for contacting us.

The antibody has been tested with rabbit samples and would be guaranteed for this species.

We guarantee all our products for TESTED species and applications as listed on the datasheet. The guarantee is valid for 6 months after a purchase. For more details about our Abpromise guarantee please check this link: https://www.abcam.com/abpromise.


I assume you will be using cultured cells or tissue sections. The following applications have been tested with this antibody: IHC-Frozen, ICC/IF (cultured cells), WB, IHC (PFA fixed).
Paraffin embedded sections have not yet been tried.

For UNTESTED species and/or applications, we have established a testing discount program. Here is a brief description of how it works:

The testing discount program is for customers who like to use an antibody/protein/kit on an untested species/application. You would purchase the antibody/protein/kit at full price, test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody/protein at the full price (100%) of the antibody/protein you have tested, or a full price discount for the amount paid for the kit. The terms and conditions applicable to this offer can be found here: https://www.abcam.com/collaborationdiscount.
This programapplies to ELISA kits and other kits we offer.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (EL4)
Loading amount
15 µg
Specification
EL4
Treatment
cells expressing recombitant viral protein
Gel Running Conditions
Reduced Denaturing
Blocking step
BSA as blocking agent for 1 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

投稿 Jan 04 2012

Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (Duodenum Biopsies)
Specification
Duodenum Biopsies
Fixative
Paraformaldehyde
Permeabilization
No
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 24°C

Mrs. Nicole Isaacs

Verified customer

投稿 May 10 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rabbit Cell lysate - whole cell (mouse hippocampal lysate)
Loading amount
5 µg
Specification
mouse hippocampal lysate
Gel Running Conditions
Reduced Denaturing (10%)
Blocking step
BSA as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Dr. Byung-il Choi

Verified customer

投稿 Feb 09 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (huh-7)
Loading amount
15000 cells
Specification
huh-7
Treatment
recombitant viral protein expressed by cell
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

投稿 Aug 04 2009

Question

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 927009 DESCRIPTION OF THE PROBLEM no bands SAMPLE Rat hippocampus PRIMARY ANTIBODY Manufacturer: Abcam Inc. Species: Rabbit Diluent: 5% milk made in tTBS Dilution: tried 1:1000, 1:500, and 1:250 Incubation time: 1 hour Wash step: washed 3 times for 15 minutes in tTBS DETECTION METHOD Amersham ECL Western Blotting Detection Reagents (GE Healthcare) POSITIVE AND NEGATIVE CONTROLS USED After we tried to probe membranes with this antibody we reprobed them with three other antibodies and got strong signals. ANTIBODY STORAGE CONDITIONS The antibody was stored at -20 C and aliquoted after the first use. SAMPLE PREPARATION Buffer: Homogenization buffer: 600ul 5M NaCl (150mM) 400ul 1M Tris pH 7.5 (20mM) 80ul 0.5M EDTA (2mM) 200ul 0.1M DTT (1mM) 200ul Triton (1%) 40ul CLAP 18.48ml H2O Protease inhibitors: CLAP: Chymostatin Leupeptin Antipain Pepstatin Heating: 5min at 95 C before loading AMOUNT OF PROTEIN LOADED About 52ug. After we tried to probe membranes with this antibody we reprobed them with three other antibodies and got strong signals. ELECTROPHORESIS/GEL CONDITIONS Non-reducing, 10% Tris/Tricine Polyacrylamide Gel TRANSFER AND BLOCKING CONDITIONS Transfer buffer: 500ml 1X Tris/Tricine/SDS Running buffer 200ml Methanol 300ml dH2O Transfer time period: 50 minutes Blocking agent: 5% milk in tTBS for 1 hour SECONDARY ANTIBODY Manufacturer: Jackson Immuno Research Species: Goat Anti-Rabbit IgG Horseradish peroxidase Diluent: 5% milk made in tTBS Dilution: tried 1:10,000 Incubation time: 1 hour Wash step: washed 3 times for 15 minutes in tTBS 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

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Answer

Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab5683 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a credit note/refund will be offered. From the protocols questionnaire, I noticed that you used 5% milk as a blocking reagent and also as diluents for the primary and secondary antibodies. I believe that the reason why you are not seeing any bands is because cross reaction between the blocking agent and antibodies might have occurred. Here at Abcam, we recommend using 5% BSA as almost all of our products have been tested with it. Please try BSA as a substitute for milk. One other possible reason why no bands were observed could also be due to insufficient antibody binding to protein of interest. A longer incubation time will ensure proper and sufficient binding. Please try incubating the primary antibody at 4oC, overnight if you have not already done so. Can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? I hope the above suggestions might prove to be useful in resolving the matter. Please try them out and should you still not get any results, please get back to me with your purchase details.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Rat Tissue sections (Sections of rat spinal cord and brain)
Specification
Sections of rat spinal cord and brain
Fixative
Paraformaldehyde

Dr. Sophie Pezet

Verified customer

投稿 Dec 19 2005

Answer

Thank you for your enquiry. Yes, ab7942 (rat brain lysate) or ab7188 (mouse brain lysate) could be used as a positive control for ab3908 and ab5683. The previous email that I sent about ab10772 [CaMKII alpha peptide - phospho T286 (phospho and non-phospho pair)] also relates to these antibodies. This peptide pair can also be used to verify that the band you see is specific. I hope this information helps, please do not hesitate to contact us if you need any more advice or information,

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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