Overview

  • Product name
    Anti-beta Actin antibody [mAbcam 8226] - Loading Control
    See all beta Actin primary antibodies
  • Description
    Mouse monoclonal [mAbcam 8226] to beta Actin - Loading Control
  • Host species
    Mouse
  • Specificity
    Does not cross-react with adult cardiac, smooth, or skeletal muscle actin. The immunogen used for this product shares 77% homology with Gamma actin/actin cytoplasmic 2. Cross-reactivity with this protein has not been confirmed experimentally.
  • Tested applications
    Suitable for: ICC/IF, ICC, Flow Cyt, IHC-FrFl, IHC-P, IHC-Fr, IP, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Horse, Chicken, Cow, Dog, Human, Pig, Zebrafish, African green monkey, Chinese hamster, Armenian hamster
    Predicted to work with: Sheep, Guinea pig
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human beta Actin.

    Read Abcam's proprietary immunogen policy (Peptide available as ab13772.)

  • Positive control
    • WB, ICC/IF, Flow Cyt: HeLa, Jurkat, A431, HEK293, NIH 3T3, PC12 cells. IHC-P: Human colon (FFPE), Rat colon (FFPE).
  • General notes

    Western blot protocol advice:

    For best results in Western blot, we recommend using 2-5% BSA for blocking not milk since we have found that using 5% milk significantly reduces the band intensity for beta actin. Please see the comparison data in the images section. If milk block is needed, we recommend using ab8224 mouse monoclonal [mAbcam 8224] to beta actin.

    Our Technical team (technical@abcam.com) will be happy to provide further information and advice.

    This antibody clone [mAbcam 8226] is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

    Abcam recommended secondaries - Goat Anti-Mouse HRP (ab205719) and Goat Anti-Mouse Alexa Fluor® 488 (ab150113).

    See other anti-mouse secondary antibodies that can be used with this antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab8226 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
ICC/IF Use a concentration of 5 µg/ml.
ICC Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

IHC-FrFl Use at an assay dependent concentration.
IHC-P Use a concentration of 0.1 - 0.5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
IHC-Fr 1/500.
IP 1/100.
WB 1/500 - 1/10000. Predicted molecular weight: 42 kDa.Can be blocked with Human beta Actin peptide (ab13772).

We recommend blocking using 2-5% BSA as we have found that use of 5% milk significantly reduces the band intensity for beta actin. Please refer to the images section for the blocking comparison data.

Target

Images

  • All lanes : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1/1000 dilution

    Lane 1 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
    Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
    Lane 4 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) (ab175783) at 1/10000 dilution

    Predicted band size: 42 kDa
    Observed band size: 42 kDa



    This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Milk before being incubated with ab8226 overnight at 4°C. Antibody binding was detected using Goat Anti-Mouse IgG H&L (Alexa Fluor® 790) (ab175783) at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

  • Immunofluorescence using ab8226 at 5µg/ml incubated for 1 hour on Rat Colon Cancer cells.

    Cells were fixed with ice-cold methanol for 5 mins, then for all following steps, permeabilised in TBS-T for 30 mins, blocked with 5% BSA for 30 mins and then washed in TBS-T. Secondary antibody was Alexa Fluor 488 goat anti-mouse IgG at 1/1000 incubated for 1 hour. Cells were counterstained with DAPI. Image at 400X magnification. All incubations were at room temperature.

    The beta actin fibres can be seen arrayed around the edge of the cells.

  • IHC image of ab8226 staining beta Actin in human colon formalin fixed paraffin embedded tissue sections*, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab8226, 0.1μg/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • IHC image of ab8226 staining beta Actin in rat colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab8226, 0.5μg/ml working concentration, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the secondary only control (shown on the inset).
    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • Lane 1 : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1/1000 dilution
    Lane 2 : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1/10000 dilution
    Lanes 3-4 : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1/500 dilution

    Lanes 1-2 : HeLa cell lysate
    Lane 3 : HEK293 cell lysate
    Lane 4 : NIH 3T3 mouse cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Rabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size: 42 kDa


    Exposure time: 10 seconds
  • All lanes : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
    Lane 3 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 4 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
    Lane 5 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 42 kDa
    Observed band size: 42 kDa


    Exposure time: 10 seconds


    Western blot image using the Optiblot Reducing Electrophoresis Kit - 10 x 10 cm (4-20%) (ab119220) with the Prism Ultra Protein Ladder (ab116028) 5µl used. We recommend using our ECL substrate kit (ab65623).

  • Lanes 1-3 : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1 µg/ml (5% BSA BLOCK)
    Lanes 4-6 : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1 µg/ml (5% MILK BLOCK)

    Lanes 1 & 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lanes 2 & 5 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
    Lanes 3 & 6 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size: 42 kDa
    Observed band size: 42 kDa


    Exposure time: 8 minutes
  • Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 0.5 µg/ml + HeLa cell lysate

    Secondary
    Goat polyclonal to mouse IgG H&L (HRP) at 1/5000 dilution

    Performed under non-reducing conditions.

    Predicted band size: 42 kDa
    Observed band size: 42 kDa


    Exposure time: 30 seconds
  • ab8226 staining beta Actin in human gastric epithelial AGS cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in formaldehyde. Permeabilization and blocking was carried out using 5% BSA containing 0.025% Triton X in TBS for 1 hour at 23°C. Primary antibody was used at 5µg/ml for 1 hour at 23°C. An Alexa Fluor 488® conjugated goat polyclonal to mouse IgG was used as the secondary antibody at a 1/1000 dilution.

    See Abreview

References

This product has been referenced in:
  • Cai XJ  et al. Effects of GABAB receptor activation on spatial cognitive function and hippocampal neurones in rat models of type 2 diabetes mellitus. Biosci Rep 38:N/A (2018). Read more (PubMed: 29176000) »
  • Han KY  et al. SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells. Genome Res 28:75-87 (2018). Read more (PubMed: 29208629) »
See all 875 Publications for this product

Customer reviews and Q&As

1-7 of 7 Q&A

Answer

Yes, F-actin is an alternative name for beta-actin, therefore the antibody will cross react. You will need to run the pull down under denaturing conditions in order to avoid pulling down actin with your NMDA receptors. A RIPA buffer would be an ideal buffer to use for this.

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Answer

You may wish to try the Anti-alpha Tubulin (ab7291) or anti-GAPDH (ab9484) but we cannot guarantee those in those conditions.

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Answer

Unfortunately, the antibodies mentioned are not suitable to detect these (human) proteins in ELISA. However, we have the following antibodies available:
1) Beta-lactoglobulin: Unfortunately, I was unable to find a human ortholog, and to my knowledge, in most experimental settings the bovine protein is detected in human breast milk (e.g. PMID8163788). If this is of interest for the customer, I am happy to let you know that we have two antibodies against the bovine beta-lactoglobulin in our catalog (ab112893, ab112894). Click here (or use the following: https://www.abcam.com/index.html?datasheet=112893). Click here (or use the following: https://www.abcam.com/index.html?datasheet=112894).
2) Alpha-lactalbumin: Again, we have antibodies against the bovine protein, but also one antibody against the human protein which are guaranteed to work in ELISA (ab112972, ab112973, ab35288). Click here (or use the following: https://www.abcam.com/index.html?datasheet=112972). Click here (or use the following: https://www.abcam.com/index.html?datasheet=112973). Click here (or use the following: https://www.abcam.com/index.html?datasheet=35288).
3) Casein: We only have one antibody against the human Casein, suitable for ELISA (ab91167). Click here (or use the following: https://www.abcam.com/index.html?datasheet=91167).
4) Lactoferrin: We have a number of antibodies available against the human lactoferrin some of which are not only guaranteed to work in a conventional ELISA, but also in Sandwich ELISA (sELISA), for example ab10110 or ab15811. Click here (or use the following: https://www.abcam.com/index.html?datasheet=10110). Click here (or use the following: https://www.abcam.com/index.html?datasheet=15811).
If the customer is interested in matched antibody pairs for Sandwich ELISA, or antibodies against these targets from other species than human, I would be pleased to assist in finding the most suitable ones.

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Answer

Indeed the same reasoning can be applied to adipocytes and we have had in the past other customers having the same problem as you. I do not know what the 25kDa band is, I would suspect it is a non specific band due to the antibody binding another non-related (or partially related) protein or it may be caused by the blocking buffer. We do not observe it in our positive controls so if you would like to investigate this further I would suggest running one of our positive controls (eg. HeLa Cell lysate),

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Answer

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

Actin is one of the most conserved eukaryotic proteins, it is expressed in mammals and birds as at least six isoforms characterized by electrophoresis and amino acid sequence analysis. There are three main actin isotypes (alpha, beta and gamma) which show >90% amino-acid (aa) homology between isotypes and >98% homology within members of a particular isotypic group. The majority of the isotype heterogeneity is located in the amino-terminal 30 aa. Four isoforms are associated with different types of muscle and two cytoskeletal isoforms that are found in virtually all non-muscle cells. The cytoskeletal isoforms are beta and gamma. The muscle isoforms are alpha (skeletal), beta (cardiac), gamma (smooth), and gamma (enteric). Beta and gamma cytoskeletal actin differ by 4 amino acids at their amino end. Our beta actin antibodies will not cross-react with adult cardiac, smooth or skeletal muscle actin. However, I suspect, depending upon the immunogen sequence used, that the beta actin antibodies could possibly cross-react with gamma actin as well.

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