Overview

  • Product nameAnti-beta Actin antibody [AC-15]
    See all beta Actin primary antibodies
  • Description
    Mouse monoclonal [AC-15] to beta Actin
  • SpecificityThis antibody makes a good loading control antibody and we recommend its use as such. We also recommend ab8226, another excellent mouse monoclonal beta actin loading control antibody.
  • Tested applicationsSuitable for: ICC/IF, Dot Blot, IHC-FoFr, ICC, IHC-P, IHC-Fr, Indirect ELISA, WB, ELISA, IHC-FrFlmore details
    Unsuitable for: IP
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Chicken, Guinea pig, Hamster, Cow, Cat, Dog, Human, Carp, Opossum, Marmoset (common), Meriones unguiculatus
    Does not react with: Drosophila melanogaster, Dictyostelium discoideum
  • Immunogen

    Synthetic peptide corresponding to beta Actin aa 1-14 (N terminal) conjugated to Keyhole Limpet Haemocyanin (KLH). Slightly modified ß-cytoplasmic actin N-terminal peptide, Ac-Asp-Asp-Asp-Ile-Ala-Al?a-Leu-Val-Ile-Asp-Asn-Gl y?-Ser-Gly-Lys, conjugated to KLH.
    Sequence:

    DDDIAALVIDNGSGK

  • EpitopeN-terminal of the beta isoform of actin.
  • Positive control
    • Cultured human or chicken fibroblasts as described in Liao et al. ICC/IF: SV40LT-SMC cells
  • General notes

    Immunofluoresence staining of chicken gizzard ultra-thin sections (North et al. J. Cell Sci. 107:445-455 (1994)) labels the dense bodies, longitudinal channels and membrane associated dense-plaque.

Properties

Applications

Our Abpromise guarantee covers the use of ab6276 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.
Dot Blot Use at an assay dependent concentration.
IHC-FoFr Use at an assay dependent concentration.
ICC 1/1000.
IHC-P 1/10000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IHC-Fr Use at an assay dependent concentration.
Indirect ELISA Use at an assay dependent concentration.
WB 1/5000 - 1/16000. Predicted molecular weight: 42 kDa.
ELISA Use at an assay dependent concentration.
IHC-FrFl Use at an assay dependent concentration.
  • Application notesIs unsuitable for IP.
  • Target

    Anti-beta Actin antibody [AC-15] images



    • Predicted band size : 42 kDa

      Lane 1: Wild-type HAP1 cell lysate (20 µg)
      Lane 2: Beta actin knockout HAP1 cell lysate (20 µg)
      Lanes 1 and 2: Merged signal (red and green). Green - ab6276 observed at 42 kDa. Red - loading control, ab181602, observed at 37 kDa.
      ab6276 was shown to specifically react with beta actin when betta actin knockout samples were used. Wild-type and beta actin knockout samples were subjected to SDS-PAGE. ab6276 and ab181602 (loading control to GAPDH) were diluted 1/5000 and 1/10 000 and incubated overnight at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

    • ab6276 staining beta Actin in SV40LT-SMC cells. The cells were fixed with 4% formaldehyde (10 min), permeabilized in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab6276 at a working concentration of 5μg/ml and ab190573, Rabbit monoclonal [EP1332Y] to alpha Tubulin (Alexa Fluor® 647, shown in red) at 1/250 overnight at +4°C, followed by a further incubation at room temperature for 1h with an anti-mouse AlexaFluor® 488 (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
      Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

    • Anti-beta Actin antibody [AC-15] (ab6276) at 1/20000 dilution + Hamster cell BHK-21 (Kidney Fibroblasts) lysate at 50000 cells

      Secondary
      IRDye 800CW-conjugated Goat anti-mouse IgG at 1/15000 dilution
      developed using the ECL technique

      Performed under reducing conditions.

      Predicted band size : 42 kDa
      Observed band size : 43 kDa (why is the actual band size different from the predicted?)


      Exposure time : 5 minutes

      This image is courtesy of an anonymous Abreview.

      Detection: Near-Infrared Fluorescence Imaging.

       



    • Predicted band size : 42 kDa

      David Grimm, Yale University, USA.

      MDCK cells induced with increasing amounts of doxycycline to control expression of the gene of interest. All cells were normalized for loading with an albumin protein standard asssay. Anti-beta actin (ab6276) was used at a concentration of 1:5000 in a milk blocking solution. B-actin blotting confirms the albumin assay in showing that an equal amount of lysate was loaded in each lane.

      MDCK cells induced with increasing amounts of doxycycline to control expression of the gene of interest. All cells were normalized for loading with an albumin protein standard asssay. Anti-beta actin (ab6276) was used at a concentration of 1:5000 in a milk blocking solution. B-actin blotting confirms the albumin assay in showing that an equal amount of lysate was loaded in each lane.

    • All lanes : Anti-beta Actin antibody [AC-15] (ab6276) at 1/5000 dilution

      Lane 1 : HeLa nuclear
      Lane 2 : HeLa whole cell lysate
      Lane 3 : A431 cell lysate
      Lane 4 : Jurkat cell lysate
      Lane 5 : HEK293 cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      Alexa Fluor anti mouse at 1/5000 dilution

      Performed under reducing conditions.

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


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

      This image is courtesy of an Abreview submitted by Dr Mark Elliott

      Western Blot of ab6276 (used as loading control) with whole tissue lysate of grey matter from BA20 (temporal cortex).  Ab6276 was diluted 1/50000 and incubated with the sample for 16 hours at 4°C.  5 µg of lysate was loaded onto the gel, which was blocked with 5% milk for 1 hour at 20°C.  An Alexa Fluor® 680 conjugated goat anti-mouse antibody, diluted 1/5000, was used as the secondary.

      Bands below actin in image are synaptophysin (SYN).

      See Abreview

    • ab6276 at 1/500 staining human macrophage cells by ICC/IF. The cells were paraformaldehyde fixed and blocked with serum prior to incubation with the antibody for 1 hour. A Cy2 ® conjugated donkey antibody was used as the secondary.

      See Abreview

    • ab6276 staining beat actin in rat hypothalamus primary cells by ICC/IF. The cells were formaldehyde fixed, permeabilized in 0.5% Triton X-100 and blocked in 5% serum for 20 minutes at 25°C. The primary antibody was diluted 1/1000 in PBS (0.1% Triton X-100, 1% goat serum) and incubated with sample for 16 hours at 4°C. An Alexa Fluor® 546 conjugated goat polyclonal to mouse IgG1, diluted 1/500 was used as secondary.

      See Abreview

    • ab6276 at 1/10000 staining mouse brain tissue sections by IHC-P. The mouse brain was immersion-fixed in 4% Formalin/PBS for 2 days, then bisected mid-saggitally. The tissue was further fixed in same solution for a further 3 days. Both halves were processed to pwax on a dip-n-dunk tissue processor using a 20hr-cycle. Sections were cut at 5microns,floated out on water at 47C, mounted on Superfrost Plus slides and dried/melted for 24hrs in a 62C oven.

      The whole brain shows positivity but there are areas/cells that are enriched. Note that the axonal tracks are negatively stained at this dilution factor, yet some cells within those tracks are still strongly positive (many similar cells outside of the tracks are also positive).

      See Abreview

    • ab6276 detecting beta actin in AGS Human gastric carcinoma cells by direct ELISA. Samples were blocked with 5% BSA for 1 hour at 23°C and incubated with the primary antibody (1/1000 in blocking buffer) for 16 hours at 4°C. Ab6729 (1/1000) was used as the secondary antibody.

      See Abreview

    References for Anti-beta Actin antibody [AC-15] (ab6276)

    This product has been referenced in:
    • Chen Y  et al. FGFR antagonist induces protective autophagy in FGFR1-amplified breast cancer cell. Biochem Biophys Res Commun N/A:N/A (2016). WB ; Human . Read more (PubMed: 26993162) »
    • Franz A  et al. Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression. Nat Commun 7:10612 (2016). Read more (PubMed: 26842564) »

    See all 506 Publications for this product

    Product Wall

    Application Western blot
    Loading amount 30 µg
    Gel Running Conditions Reduced Denaturing (12)
    Sample Human Tissue lysate - whole (CNS tissue)
    Specification CNS tissue
    Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Username

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    Verified customer

    Submitted Aug 01 2014

    Application Immunocytochemistry/ Immunofluorescence
    Blocking step BSA as blocking agent for 45 minute(s) · Concentration: 2% · Temperature: RT°C
    Sample Human Cell (HeLa cells)
    Specification HeLa cells
    Permeabilization Yes - 0.2% Triton-X100
    Fixative Paraformaldehyde
    Username

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    Submitted May 23 2014

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Western blot
    Loading amount 25 µg
    Gel Running Conditions Reduced Denaturing (12.5%)
    Sample Mouse Cell lysate - whole cell (Mouse brain lysates)
    Specification Mouse brain lysates
    Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
    Username

    Herr Dr. Vladimir Milenkovic

    Verified customer

    Submitted Jul 10 2013

    Abcam has not validated the combination of species/application used in this Abreview.
    Application Western blot
    Sample Dog Cell lysate - whole cell (Canine Kidney Epithelial MDCK cells)
    Gel Running Conditions Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)
    Loading amount 50000 cells
    Specification Canine Kidney Epithelial MDCK cells
    Blocking step Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
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    Submitted Sep 23 2016

    Abcam has not validated the combination of species/application used in this Abreview.
    Application Western blot
    Sample African Green Monkey Cell lysate - whole cell (VERO E6)
    Gel Running Conditions Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)
    Loading amount 75000 cells
    Specification VERO E6
    Blocking step No blocking step used for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
    Username

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    Submitted Jun 28 2016

    Abcam has not validated the combination of species/application used in this Abreview.
    Application Western blot
    Sample Hamster Tissue lysate - whole (BHK-21 (Kidney Fibroblasts))
    Gel Running Conditions Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)
    Loading amount 50000 cells
    Specification BHK-21 (Kidney Fibroblasts)
    Blocking step Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
    Username

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    Verified customer

    Submitted Jun 19 2016

    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application Western blot
    Sample Rat Tissue lysate - whole (spinal cord dorsal horn)
    Gel Running Conditions Reduced Denaturing (15%)
    Loading amount 30 µg
    Specification spinal cord dorsal horn
    Blocking step 1x Rapid block as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
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    Submitted Mar 03 2016

    Application Western blot
    Sample Human Cell lysate - whole cell (Human Foreskin Fibroblasts)
    Gel Running Conditions Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)
    Loading amount 100000 cells
    Specification Human Foreskin Fibroblasts
    Blocking step Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 24 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 4°C
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    Submitted Jan 20 2016

    Application Western blot
    Sample Mouse Cell lysate - whole cell (mouse embryonic fibroblasts)
    Gel Running Conditions Reduced Denaturing
    Loading amount 10000 cells
    Treatment With or without 100uM zVAD
    Specification mouse embryonic fibroblasts
    Blocking step Li-Cor Odyssey Blocking Buffer (TBS) as blocking agent for 14 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 4°C
    Username

    Dr. Yi-Chieh Perng

    Verified customer

    Submitted Nov 30 2015

    Application Immunocytochemistry/ Immunofluorescence
    Sample Chinese Hamster Cell (CHO cell)
    Permeabilization Yes - 1% TRITON-X-100
    Specification CHO cell
    Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C
    Fixative Paraformaldehyde
    Username

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    Submitted Nov 26 2015

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