使用敲除细胞株进行验证

Anti-beta Actin抗体[AC-15] (ab6276)

概述

  • 产品名称Anti-beta Actin抗体[AC-15]
    参阅全部 beta Actin 一抗
  • 描述
    小鼠单克隆抗体[AC-15] to beta Actin
  • 特异性This 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.
  • 经测试应用适用于: ICC/IF, IHC-FoFr, ICC, IHC-P, IHC-Fr, Indirect ELISA, WB, ELISAmore details
  • 种属反应性
    与反应: Mouse, Rat, Sheep, Rabbit, Chicken, Guinea pig, Hamster, Cow, Cat, Dog, Human, Carp, Opossum, Common marmoset, Meriones unguiculatus
    不与反应: Drosophila melanogaster, Dictyostelium discoideum
  • 免疫原

    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

  • 表位N-terminal of the beta isoform of actin.
  • 阳性对照
    • Cultured human or chicken fibroblasts as described in Liao et al. ICC/IF: SV40LT-SMC cells
  • 常规说明

    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.

    This product was changed from ascites to tissue culture supernatant on 31st January 2017. The following lots are from ascites and are still in stock on 31st January 2017- GR231981, GR247612 and GR181659. Lot numbers higher than GR247612 will be from tissue culture supernatant. Please note that the dilutions may need to be adjusted accordingly.

性能

应用

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.

应用 Ab评论 说明
ICC/IF Use a concentration of 5 µg/ml.
IHC-FoFr Use at an assay dependent concentration.

Use at an assay dependent concentration.

ICC Use at an assay dependent concentration.

Use at an assay dependent concentration.

IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Use at an assay dependent concentration.

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.

靶标

Anti-beta Actin antibody [AC-15] 图像



  • 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-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) 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).

  • 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 47°C, mounted on Superfrost Plus slides and dried/melted for 24hrs in a 62°C 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 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

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

    Secondary
    Goat anti-mouse IgG H&L (IRDye® 800CW) 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.

     

    See Abreview



  • Predicted band size : 42 kDa

    This image is courtesy of 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 assay. 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.

  • 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

  • Anti-beta Actin antibody [AC-15] (ab6276) at 1/20000 dilution + Rabbit Reticulocyte whole cell lysate at 0.5 µg

    Secondary
    Goat anti-mouse IgG H&L (IRDye® 680RD) at 1/15000 dilution

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


    Exposure time : 2 minutes

    This image is courtesy of an anonymous abreview.

    Reduced, denatured 4-12% Bolt Bis-Tris Plus gel. 
    Blocking: 100% Li-Cor Odyssey Blocking Buffer (TBS).

    See Abreview



  • 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 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

Anti-beta Actin antibody [AC-15] (ab6276)参考文献

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 926 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
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提交于 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
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提交于 May 23 2014

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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
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Herr Dr. Vladimir Milenkovic

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提交于 Jul 10 2013

Application Western blot
Sample Chinese Hamster Cell lysate - whole cell (Chinese hamster ovary (CHO) cells)
Gel Running Conditions Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)
Loading amount 50000 cells
Specification Chinese hamster ovary (CHO) 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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提交于 Dec 12 2016

Abcam has not validated the combination of species/application used in this Abreview.
Application Western blot
Sample Rabbit Cell lysate - whole cell (Rabbit Reticulocyte)
Gel Running Conditions Reduced Denaturing (4-12% Bolt Bis-Tris Plus gel)
Loading amount 0.5 µg
Specification Rabbit Reticulocyte
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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提交于 Nov 21 2016

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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提交于 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
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提交于 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
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提交于 Jun 19 2016

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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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提交于 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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提交于 Jan 20 2016

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