Overview

  • Product name
    Anti-Sarcomeric Alpha Actinin antibody [EA-53]
    See all Sarcomeric Alpha Actinin primary antibodies
  • Description
    Mouse monoclonal [EA-53] to Sarcomeric Alpha Actinin
  • Host species
    Mouse
  • Specificity
    According to the following publication, this antibody clone detects the Alpha Actinin 2. However, we have not confirmed this with internal testing if it detects Actinin 1 or 2. https://academic.oup.com/hmg/article/14/12/1587/640357/Alpha-actinin-associates-with-polycystin-2-and
  • Tested applications
    Suitable for: IHC-P, IHC-Fr, ICC/IF, IHC-FoFr, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Goat, Chicken, Hamster, Cow, Cat, Dog, Human, Pig, Xenopus laevis, Fish, Lizard, Snake, Xenopus tropicalis
  • Immunogen

    Full length native protein (purified) corresponding to Rabbit Sarcomeric Alpha Actinin. Purified rabbit skeletal α-actinin.

  • Positive control
    • WB: Mouse and rat skeletal muscle and rat heart tissue lysates. IHC-P: Human skeletal muscle tissue.
  • General notes

    Abcam is committed to meeting high standards of ethical manufacturing and has decided to discontinue this product by June 2019 as it has been generated by the ascites method. We are sorry for any inconvenience this may cause. We would recommend antibody ab68167 as a replacement.

Properties

Applications

Our Abpromise guarantee covers the use of ab9465 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 1/25 - 1/50. ABC method. We suggest an incubation period of 60 minutes at room temperature. However, depending upon the fixation conditions and the staining system employed, optimal incubation and dilutions should be determined by the user.
IHC-Fr Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration. PubMed: 17383364
IHC-FoFr Use at an assay dependent concentration. PubMed: 20019242
WB Use a concentration of 1 µg/ml. Detects a band of approximately 103 kDa (predicted molecular weight: 103 kDa).

Target

  • Function
    F-actin cross-linking protein which is thought to anchor actin to a variety of intracellular structures. This is a bundling protein.
  • Tissue specificity
    Expressed in both skeletal and cardiac muscle.
  • Involvement in disease
    Defects in ACTN2 are the cause of cardiomyopathy dilated type 1AA (CMD1AA) [MIM:612158]. Dilated cardiomyopathy is a disorder characterized by ventricular dilation and impaired systolic function, resulting in congestive heart failure and arrhythmia. Patients are at risk of premature death.
  • Sequence similarities
    Belongs to the alpha-actinin family.
    Contains 1 actin-binding domain.
    Contains 2 CH (calponin-homology) domains.
    Contains 2 EF-hand domains.
    Contains 4 spectrin repeats.
  • Cellular localization
    Cytoplasm > myofibril > sarcomere > Z line. Colocalizes with MYOZ1 and FLNC at the Z-lines of skeletal muscle.
  • Information by UniProt
  • Database links
  • Alternative names
    • Actin binding protein antibody
    • Actinin alpha 2 antibody
    • ACTN 2 antibody
    • ACTN2 antibody
    • ACTN2_HUMAN antibody
    • Alpha actinin 2 antibody
    • Alpha actinin skeletal muscle antibody
    • Alpha actinin skeletal muscle isoform 2 antibody
    • Alpha-actinin skeletal muscle isoform 2 antibody
    • Alpha-actinin-2 antibody
    • CMD1AA antibody
    • F actin cross linking protein antibody
    • F-actin cross-linking protein antibody
    see all

Images

  • ab9465 staining Sarcomeric Alpha Actinin in mouse heart tissue by Immunohistochemistry (Frozen sections). Serial longitudinal (12 µm) sections from wt hearts were incubated with ab9465. Nuclei were visualized with DAPI.
  • Immunocytochemistry/Immunofluorescence analysis of H9 hESC and CBiPSC6.2 cells labelling Sarcomeric Alpha Actinin (green) with ab9465 at a dilution of 1/200. An Alexa Fluor® 488-conjugated goat anti-mouse IgG was used as the secondary antibody (1/200).

  • Immunofluorescence staining of SNAIL1 and cardiac cell type markers (alpha actinin-cardiomyocyte marker; 1/100 dilution) in the ventricular infarct zone of LAD surgery mice. Scale bars: 50 microns.

    5 um sections of paraffin or OCT embedded hearts were utilized for Massons Trichrome Staining and immunofluorescence. For SNAIL1 immunofluorescence, antigen retrieval was done in Nuclear Decloaker solution and TSA plus kit was used to amplify SNAIL1 signal.

  • All lanes : Anti-Sarcomeric Alpha Actinin antibody [EA-53] (ab9465) at 1/250 dilution

    Lane 1 : Mouse skeletal muscle tissue lysate
    Lane 2 : Rat skeletal muscle tissue lysate
    Lane 3 : Rat heart tissue lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 103 kDa
    Observed band size: 103 kDa
    Additional bands at: 25 kDa, 57 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 90 seconds
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skeletal muscle tissue staining Sarcomeric Alpha Actinin with ab9465.

References

This product has been referenced in:
  • Sabblah TT  et al. A novel mouse model carrying a human cytoplasmic dynein mutation shows motor behavior deficits consistent with Charcot-Marie-Tooth type 2O disease. Sci Rep 8:1739 (2018). IHC . Read more (PubMed: 29379136) »
  • Sun AJ  et al. Comparison of mouse brown and white adipose-derived stem cell differentiation into pacemaker-like cells induced by TBX18 transduction. Mol Med Rep 17:7055-7064 (2018). Read more (PubMed: 29568953) »
See all 71 Publications for this product

Customer reviews and Q&As

1-10 of 22 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Sheep Tissue sections (Muscle)
Permeabilization
Yes - 0,2 % Triton-X in PBS
Specification
Muscle
Fixative
Methanol:Acetone 1:1

Kassandra Walluks

Verified customer

Submitted Apr 24 2018

Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (Heart)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate Buffer
Permeabilization
Yes - 0.2% Triton X-100
Specification
Heart
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 26 2016

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Cardiac cells)
Specification
Cardiac cells
Blocking step
Serum as blocking agent for 10 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Sep 16 2016

Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (Heart)
Antigen retrieval step
None
Permeabilization
Yes - Triton x-100, 0.01%
Specification
Heart
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 05 2015

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (heart)
Permeabilization
Yes - Triton x-100, 0.01%
Specification
heart
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jun 17 2015

Application
Western blot
Loading amount
25 µg
Gel Running Conditions
Reduced Denaturing
Sample
Rat Tissue lysate - whole (heart)
Specification
heart
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Dr. Shyam Sundar Nandi

Verified customer

Submitted Nov 25 2014

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1%
Sample
Rat Cell (Heart)
Specification
Heart
Permeabilization
Yes - Triton x-100, 0.01%
Fixative
Paraformaldehyde

Dr. Shyam Sundar Nandi

Verified customer

Submitted Nov 20 2014

Answer

Thank you for contacting us. Because we carry over 70,000 products, it isn't feasible for us to keep small sample sizes of our products.

We are happy to reassure our customers that all of our products are covered by our Abpromise, which guarantees that the product will work in the applications and species specified on the datasheet, or we will offer a replacement, credit, or refund within 6 months of purchase.

In addition, we have a Polymer IHC Kit which tackles exactly the problem of a high background which arises from this kind of staining (mouse-on-mouse staining):

https://www.abcam.com/index.html?datasheet=127055 (or use the following: https://www.abcam.com/index.html?datasheet=127055).

ab127055 allows the use of mouse antibodies on mouse tissue to obtain excellent signal intensities and clean background.

Another alternative i could recommend would be using three antibodies in total: Use a Fab-fragment anti-mouse antibody against the H+L chain to block the endogenous mouse IgGs, than incubate with your primaries and (as you suggested) use an isotype specific secondary antibody.

A Fab fragment antibody has the advantage that it can "cover" the endogenous mouse IgG tighter than a whole IgG antibody.

https://www.abcam.com/index.html?datasheet=6668 (or use the following: https://www.abcam.com/index.html?datasheet=6668).



I hope this information is helpful. Please do not hesitate to contact us again with any other questions.

Read More

Question
Answer

Thank you for your call yesterday and for your patience while I've looked into your enquiry.

I've heard back from the lab, and there is 0.05% sodium azide in this antibody. I will update the datasheet and have an MSDS madepromptly. Please let me know if you have any further questions or if there is anything else that we can do for you, and I'll be happy to help.

Read More

Question


Thank you for help about this case.
Attached please find the detail and data.
Would you please help deal with this issue?

Thank you very much.
Best regards,

Iris

1) Abcam product code ab9465

2) Abcam order reference number or product batch number GR50514-3

3) Description of the problem
1. ICC for cardiomyocytes, striated form was not appeared.
2. Non-specific binding for 3T3 fibroblasts.
4) Sample preparation:
Species mouse
Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other: cells in culture (cardiomyocytes primary culture/ 3T3 fibroblasts)
Sample preparation
Positive control
Negative control

5) Fixation step
Yes/No Yes
If yes: Fixative agent and concentration 3.7% PFA in PBS
Fixation time 20 min
Fixation temperature 4℃
6) Antigen retrieval method No

7) Permeabilization method:
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers? Yes, 15 min, room temp.
Permeabilizing agent and concentration: 0.1% Triton X-100 in PBS

8) Blocking agent (eg BSA, serum…):
Concentration 2%
Blocking time 20 min
Blocking temperature room temp.

9) Endogenous peroxidases blocked? no
Endogenous biotins blocked? no
10) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution 1:100
Diluent buffer 2% BSA in 0.1% PBST
Incubation time 1 hr room temp.


11) Secondary antibody:
Species: rabbit
Reacts against: Mouse
Concentration or dilution 1 hr
Diluent buffer 2% BSA in 0.1% PBST
Incubation time Fluorochrome or enzyme conjugate FITC

12) Washing after primary and secondary antibodies:
Buffer PBS,
Number of washes three˜five times, 5 mins
13) Detection method microscope

14) How many times have you run this staining? 5 times
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this antibody? No

Read More
Answer

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.

I apologize for the inconvenience and am pleased to offer you a free of charge replacement or credit note in compensation.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Read More

1-10 of 22 Abreviews or Q&A

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

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