Overview

  • Product name
    Anti-Actin antibody [AC-40]
    See all Actin primary antibodies
  • Description
    Mouse monoclonal [AC-40] to Actin
  • Host species
    Mouse
  • Tested applications
    Suitable for: Dot blot, WB, ELISA, IHC-FoFr, IHC-P, IHC-Fr, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Goat, Chicken, Guinea pig, Hamster, Cow, Dog, Human, Pig, Xenopus laevis, Carp, Snail
  • Immunogen

    Synthetic peptide within Human Actin aa 365-375 (C terminal). The exact sequence is proprietary.
    Sequence:

    SGPSIVHRKCF

  • Epitope
    Monoclonal anti-Actin recognizes an epitope located on the C-terminal end of actin. This epitope is conserved in all actin isoforms.
  • General notes

    Storage in frost-free freezers is not recommended. If slight turbidity occurs upon prolonged storage, clarify the solution by centrifugation before use.

    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 ab219733 as a replacement.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Applications

Our Abpromise guarantee covers the use of ab11003 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
Dot blot Use at an assay dependent concentration.

Use at an assay dependent concentration

WB Use at an assay dependent concentration. Predicted molecular weight: 42 kDa.

1/500 using cultured Human or chicken fibroblast extract. Predicted molecular weight: 42 kDa.

ELISA Use at an assay dependent concentration.
IHC-FoFr Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.

1/200 determined by indirect immunofluorescent staining of cultured Human or chicken fibroblasts.

Target

  • Function
    Actins are highly conserved proteins that are involved in various types of cell motility and are ubiquitously expressed in all eukaryotic cells.
  • Involvement in disease
    Defects in ACTA1 are the cause of nemaline myopathy type 3 (NEM3) [MIM:161800]. A form of nemaline myopathy. Nemaline myopathies are muscular disorders characterized by muscle weakness of varying severity and onset, and abnormal thread-or rod-like structures in muscle fibers on histologic examination. The phenotype at histological level is variable. Some patients present areas devoid of oxidative activity containg (cores) within myofibers. Core lesions are unstructured and poorly circumscribed.
    Defects in ACTA1 are a cause of myopathy congenital with excess of thin myofilaments (MPCETM) [MIM:161800]. A congenital muscular disorder characterized at histological level by areas of sarcoplasm devoid of normal myofibrils and mitochondria, and replaced with dense masses of thin filaments. Central cores, rods, ragged red fibers, and necrosis are absent.
    Defects in ACTA1 are a cause of congenital myopathy with fiber-type disproportion (CFTD) [MIM:255310]; also known as congenital fiber-type disproportion myopathy (CFTDM). CFTD is a genetically heterogeneous disorder in which there is relative hypotrophy of type 1 muscle fibers compared to type 2 fibers on skeletal muscle biopsy. However, these findings are not specific and can be found in many different myopathic and neuropathic conditions.
  • Sequence similarities
    Belongs to the actin family.
  • Cellular localization
    Cytoplasm > cytoskeleton.
  • Information by UniProt
  • Database links
  • Alternative names
    • a actin antibody
    • ACTA antibody
    • ACTA1 antibody
    • Actin alpha skeletal muscle antibody
    • Actin antibody
    • actin, alpha 1, skeletal muscle 1 antibody
    • actin, alpha 1, skeletal muscle antibody
    • Actin, alpha skeletal muscle antibody
    • actina antibody
    • actine antibody
    • ACTS_HUMAN antibody
    • aktin antibody
    • Alpha Actin 1 antibody
    • Alpha skeletal muscle Actin antibody
    • alpha skeletal muscle antibody
    • alpha-actin antibody
    • Alpha-actin-1 antibody
    • ASMA antibody
    • CFTD antibody
    • CFTD1 antibody
    • CFTDM antibody
    • MPFD antibody
    • NEM1 antibody
    • NEM2 antibody
    • NEM3 antibody
    • nemaline myopathy type 3 antibody
    see all

Images

  • ab11003 staining Actin in Human SW480 cells by Immunocytochemistry/ Immunofluorescence. The cells were formaldehyde fixed and then blocked using 10% serum for 1 hour at 25°C. Samples were then incubated with primary antibody at 1/50 for 2 hours at 25°C. The secondary antibody used was a goat anti-mouse IgG conjugated to Alexa Fluor® 568 (red) used at a 1/100 dilution.

    See Abreview

  • Immunohistochemical analysis of frozen Human tongue tissue, using ab11003.

  • ab11003 staining Human fibroblast cells by ELISA (Sandwich-capture). Cells were blocked in 1% BSA for 1 hour at 25°C. The  primary antibody ab11003 (Capture antibody) was diluted 1/3000 and incubated with sample for 12 hours at 4°C. ab5694 was used as secondary and a HRP conjugated goat polyclonal to mouse, diluted 1/1000 was used as detection antibody.

    See Abreview

References

This product has been referenced in:
  • Shi R  et al. Nanosphere-mediated co-delivery of VEGF-A and PDGF-B genes for accelerating diabetic foot ulcers healing in rats. Gene Ther 25:425-438 (2018). Read more (PubMed: 29955127) »
  • Liaunardy-Jopeace A  et al. Encoding optical control in LCK kinase to quantitatively investigate its activity in live cells. Nat Struct Mol Biol 24:1155-1163 (2017). WB . Read more (PubMed: 29083415) »
See all 28 Publications for this product

Customer reviews and Q&As

1-7 of 7 Q&A

Question

I have a problem with the antibody ab6276. I try to develop an indirect ELISA with this antibody, but do not get any result. This is the procedure, I've performed: - Coating of ELISA plate (Costar high bind) with actin (rabbit skeletal muscle, Cytoskeleton, inc. # AKL95)in carbonate/ bicarbonate buffer pH 9.6. Concentration range: 0.625 - 10 µg/mL), coating for 1h at room temperature or over night at 4 ˚C - Blocking: 5 % milk in PBST, 1h at room temperature - Detection antibody (ab6276): dilution in blocking buffer (see above), incubation for 1 h at room temp, dilution: 1:500 - 1:4000 - Secondary antibody (goat anti-mouse IgG HRP, santa cruz biotechnology, # sc-2005), dilution in blocking buffer (see above), incubation for 1 h at room temp, dilution: 1:2000 - 1:8000 - Detection with TMB substrate. No color development, also not after 2 hours. Between all steps, the plate was washed 3x with TBST (200 µL wash buffer/ well) In order to find the problem, I did the following tests: Antigen adsorption study with BCA assay. Antigen did adsorb on surface with a concentration of about 1 µg/mL. Adsorption of secondary antibody on plate, detection with secondary antibody: positive, ABs react with each other. Test of secondary AB with substrate: positive. Increasing the secondary antibody concentration to 1:1000, color development was detected, but the negative control (no antigen) showed the same intensity like the samples. SDS PAGE/ Western blot with actin from the same source: positive. Do you have any suggestions? My thought is, that the protein is maybe folded in a way, that the antibody can't bind to the epitope since it is able to bind to the denatured protein in the western blot.

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Answer

Thank you for contacting us.

I would not recommend adding SDS or a reducing agent to the sample prior to coating, as this may interfere with the coating efficiency. It is possible that by boiling the protein for 5-10 minutes, the heat denaturation may be enough to expose the epitope that is being recognized by this antibody in WB.

This antibody is designed for detection of beta actin, not skeletal muscle actin. The homology between rabbit skeletal muscle actin (http://www.uniprot.org/uniprot/P68135)and the immunogen for this antibody is only 64%. Using an antibody directed against a more conserved region of actin, such as ab11003, may give better results. This antibody shows 100% homology with the above mentioned rabbit sequence.

I hope this helps, please let me know if you need any additional information or assistance.

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Question
Answer

The new lot of ab11003 is currently in transit from the production lab to our stock and there is no lot number allocated yet, but I can confirm that this new lot is different from the lot your customer has received originally.

I will send you the lot number as soon as it is created.

We do not have set expiration dates for our products.Aliquotted, not diluted and stored at -20ºC, antibodies, includingab11003is stable for up to 5 years. Up to 10 years when stored at -80ºC. It is very importantto avoid repeated freeze / thaw cycles.We guarantee all of our products to work for at least 120 days from the date of purchase when stored correctly.For more information on antibody storage and stability, please visit our Antibody Storage Guide :https://www.abcam.com/index.html?pageconfig=resource&rid=10795 .

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Answer

Thank you for your reply.

Unfortunately we do not have any other lot of ab11003 in stock.

Would your customer be interested in trying an alternative anti-actin from the following list (excluding ab11003 of course) : https://www.abcam.com/index.html?pageconfig=searchresults&search=Actin&pt=1&sk=conj&sv=0&sn=Unconjugated&l=2&fViewMore=1

Thank you.

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Question

Resend
1) Abcam product code ab11003

2) Abcam order reference number or product batch number

Cat no:AB11003

Lot no.: GR2058-9

3) Description of the problem: no band / high background (many non-specific bands) / wrong band size / other? Some are high background; some have no bands

4) Sample preparation:

Type of sample (whole cell lysates, fraction, recombinant protein…): brain lysate

Species : mice

Lysis buffer : RIPA

Protease inhibitors: Roche complete

Phosphatase inhibitors : Na-β-glycerophosphate, Na Orthovanadate

Reducing agent : b-mercapethanol

Boiling for ≥5 min? yes

Protein loaded ug/lane or cells/lane : 250

Positive control : c57 mouse brain lysate; Cre-transfected HEK

Negative control : HEK





5) Percentage of gel

Type of membrane: SDS-PAGE 10%

Protein transfer verified : Yes, all the ladders pass through

Blocking agent and concentration 5% BSA

Blocking time 1 hr

Blocking temperature Room Temperature





6) Primary antibody (If more than one was used, describe in “additional notes”) :

Concentration or dilution 1:1000

Diluent buffer : 5% BSA

Incubation time Overnight

Incubation temperature: 4 deg C



7) Secondary antibody:

Species: Goat

Reacts against: Mouse

Concentration or dilution 1:5000

Diluent buffer 1% BSA in PBST-0.1%

Incubation time 1 hour

Incubation temperature: Room Temperature

Fluorochrome or enzyme conjugate: HRP





8) Washing after primary and secondary antibodies:

Buffer PBST-0.1%;

Number of washes 3 times



9)Detection method: 1 Super Signal West Pico Stable Peroxide Solution : 1mL Enhancer Solution or

Femto







10) How many times have you run this staining?

Do you obtain the same results every time? 7; 2 by other colleagues
What steps have you altered to try and optimize the use of this antibody? I changed lysis buffer and added in c57 mice and HEK cell for comparison. Actin antibody was the last thing I thought could be wrong until it was verified by other colleagues.

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Answer

Thank you for filling in the questionnaire and for reporting this problem to us. We will use the details you sent about your protocol in order to investigate the source of the problem.

I have looked through your protocol, which looks fine to me. It seems that you have received a faulty tube or lot of ab11003. I appreciate the time that has been spent on these experiments and would be pleased to arrange in compensation a credit note or areplacement with an alternative anti-actin from our catalog. The link to thelist of Abcamanti-actin antibodies is : https://www.abcam.com/index.html?pageconfig=searchresults&search=Actin&pt=1&sk=conj&sv=0&sn=Unconjugated&l=2&fViewMore=1

I look forward to hearing from you with details of how you andyour customerwould like to proceed.

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Answer

Thank you for contacting us.

I am sorry to hear your customeris experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.

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Answer

Thank you for your enquiry. Ab8226 does not cross-react with ADULT cardiac, smooth, or skeletal muscle actin. Myoblasts are immature muscle cells and looking through the literature it does seem that some people use actin as a loading control. Your customer may want to try a tubulin loading control. However, I do not want to recommend a product that is unsuitable and suggest that you refer to the latest literature in this field.

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Question
Answer

Thank you for your email. Ab11003 is in unpurified form, ascites, and so we are unable to provide a concentration. If you have any more questions, please let us know.

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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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