Overview

  • Product name
    Anti-CaMKII (phospho T286) antibody
    See all CaMKII primary antibodies
  • Description
    Rabbit polyclonal to CaMKII (phospho T286)
  • Host species
    Rabbit
  • Specificity
    This antibody is specific for the ~50 kDa alpha CaM Kinase II subunit and the ~60 kDa beta CaM Kinase II subunit phosphorylated at Thr286 in Western blots. Immunolabeling is blocked by the phosphopeptide used as the antigen but not by the corresponding dephosphopeptide.
  • Tested applications
    Suitable for: WB, IHC-P, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Pig, Xenopus laevis
    Predicted to work with: Chicken, Cow
  • Immunogen

    Synthetic phosphopeptide corresponding to amino acids surrounding phospho-286 from Rat brain CaMKII.

  • Positive control
    • Rat cortex lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab32678 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 1/1000. Predicted molecular weight: 50 kDa. Predicted molecular weight: ~50 kDa for the alpha subunit and ~60 kDa for the beta subunit of CaMKII.
IHC-P Use a concentration of 2 µg/ml.
IHC-Fr Use at an assay dependent concentration.

Target

Images

  • All lanes : Anti-CaMKII (phospho T286) antibody (ab32678) at 1/1000 dilution

    Lane 1 : Human myocardium tissue lysate, heart failure
    Lane 2 : Human myocardium tissue lysate, hypertrophy
    Lane 3 : Human myocardium tissue lysate, non-failing

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : HRP conjugated sheep anti-rabbit at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 50 kDa
    Observed band size: 50 kDa


    Exposure time: 30 seconds


    Blocked with 5% milk for 1 hour at RT.

    Incubated with primary antibody for 14 hours at 4°C in TBS-T20.

    See Abreview

  • ab32678 (2µg/ml) staining CaMKII (phospho T286) in human Brain:cortex:frontal-lateral using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of nuclear/cytoplasmic compartments within the stellate cells and the myelinated fibres of white matter region .
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

  • PC 12 cells were incubated at 37°C for 30 minutes with vehicle control (0 µM) and different concentrations of myricetin (ab120721). Increased expression of CaMKII (phospho T286) (ab32678) in PC 12 cells correlates with an increase in myricetin concentration, as described in literature.

    Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 3% milk before being incubated with ab32678at 1/500 dilution and ab52476 at 1/500 dilution overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

  • All lanes : Anti-CaMKII (phospho T286) antibody (ab32678) at 1/1000 dilution

    Lane 1 : Rat brain cortex lysate.
    Lane 2 : Rat brain cortex lysate preincubated with lambda-phosphatase.

    Lysates/proteins at 50 µg per lane.

    Predicted band size: 50 kDa
    Observed band size: 50,60 kDa
    why is the actual band size different from the predicted?



    Predicted molecular weight: ~50 kDa for the alpha subunit and ~60 kDa for the beta subunit of CaMKII.
    These images show the phosphospecificity of this antibody.

References

This product has been referenced in:
  • Misrani A  et al. Differential effects of citalopram on sleep-deprivation-induced depressive-like behavior and memory impairments in mice. Prog Neuropsychopharmacol Biol Psychiatry 88:102-111 (2019). Read more (PubMed: 30017777) »
  • Zhong W  et al. Intracellular emetic signaling cascades by which the selective neurokinin type 1 receptor (NK1R) agonist GR73632 evokes vomiting in the least shrew (Cryptotis parva). Neurochem Int 122:106-119 (2019). Read more (PubMed: 30453005) »
See all 44 Publications for this product

Customer reviews and Q&As

1-10 of 31 Abreviews or Q&A

Application
Western blot
Sample
Mouse Cell lysate - whole cell (BV2 microglia)
Gel Running Conditions
Reduced Denaturing
Loading amount
50 µg
Treatment
5µM LPA for different time points
Specification
BV2 microglia
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Dr. Lisha Joshi

Verified customer

Submitted Jun 19 2019

Application
Western blot
Sample
Rat Cell lysate - whole cell (primary culture cortex cell)
Gel Running Conditions
Non-reduced Denaturing (15% SDS-PAGE)
Loading amount
15 µg
Treatment
RIPA & 1%Protease inhibitor for 15min on ice
Specification
primary culture cortex cell
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Oct 08 2018

Application
Immunohistochemistry (Frozen sections)
Sample
Pig Tissue sections (Left ventricle)
Permeabilization
No
Specification
Left ventricle
Blocking step
(agent) for 10 minute(s) · Concentration: 100% · Temperature: 25°C
Fixative
Acetone

Christopher Trummer

Verified customer

Submitted Oct 02 2018

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Left ventricle)
Permeabilization
No
Specification
Left ventricle
Blocking step
(agent) for 10 minute(s) · Concentration: 100% · Temperature: 25°C
Fixative
Acetone

Christopher Trummer

Verified customer

Submitted Oct 02 2018

Application
Western blot
Sample
Mouse Tissue lysate - whole (Cortex)
Gel Running Conditions
Reduced Denaturing (4-12%)
Loading amount
50 µg
Specification
Cortex
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Christopher Trummer

Verified customer

Submitted Oct 02 2018

Application
Western blot
Sample
Guinea pig Tissue lysate - whole (Left ventricle)
Gel Running Conditions
Reduced Denaturing (4-12%)
Loading amount
50 µg
Specification
Left ventricle
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Christopher Trummer

Verified customer

Submitted Oct 02 2018

Application
Western blot
Sample
Pig Tissue lysate - whole (Left ventricle)
Gel Running Conditions
Reduced Denaturing (4-12%)
Loading amount
50 µg
Specification
Left ventricle
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Christopher Trummer

Verified customer

Submitted Oct 02 2018

Application
Western blot
Sample
Human Tissue lysate - whole (Left ventricle)
Gel Running Conditions
Reduced Denaturing (4-12%)
Loading amount
50 µg
Specification
Left ventricle
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Christopher Trummer

Verified customer

Submitted Oct 02 2018

Application
Western blot
Sample
Rabbit Tissue lysate - whole (Cortex)
Gel Running Conditions
Reduced Denaturing
Loading amount
50 µg
Specification
Cortex
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Christopher Trummer

Verified customer

Submitted Oct 02 2018

Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (Left ventricle)
Permeabilization
No
Specification
Left ventricle
Blocking step
(agent) for 10 minute(s) · Concentration: 100% · Temperature: 25°C
Fixative
Acetone

Christopher Trummer

Verified customer

Submitted Oct 02 2018

1-10 of 31 Abreviews or Q&A

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