Anti-beta Actin antibody [mAbcam 8226] (ab8226)

Overview

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). Abcam recommends blocking and incubation using 5% BSA as we have found that use of 5% milk significantly reduces the intensity of the signal obtained. Please refer to WB image for further details.

Target

Anti-beta Actin antibody [mAbcam 8226] images

  • 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.

  • 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

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

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

    Lysates/proteins at 10 µg per lane.

    Secondary
    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
  • All lanes : Anti-beta Actin antibody [mAbcam 8226] (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
    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 a goat anti-mouse Alexa 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 5ug/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.

  • All lanes : Anti-beta Actin antibody [mAbcam 8226] (ab8226) at 1 µg/ml

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

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/50000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

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


    Exposure time : 3 minutes

    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 2% Bovine Serum Albumin before being incubated with ab8226 overnight at 4°C. Antibody binding was detected using an anti-mouse HRP (ab97040) antibody conjugated to HRP, and visualised using ECL development solution ab133406

  • ab8226 staining beta Actin in human AGS cells by Immunocytochemistry. Cells were fixed with formaldehyde, permeabilized with 0.025% Triton-X in TBS and blocking with 5% serum was performed for 1 hour at 230C. Samples were incubated with primary antibody (1/500) for 1 hour at 23°C. An HRP®-conjugated goat polyclonal to mouse IgG was used undiluted as secondary antibody.

    See Abreview

  • Anti-beta Actin antibody [mAbcam 8226] (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

  • All lanes : Anti-beta Actin antibody [mAbcam 8226] (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
    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).
    Loading Control (ab8226) used at 1 µg/ml.

    Lane 1 : Hela cell lysate
    Lane 2 : Jurkat cell lysate
    Lane 3 : A431 cell lysate
    Lane 4 : HEK293 cell lysate
    Lane 5 : HepG2 cell lysate

    Lysates loaded at 20 µg per lane.

     

    Secondary antibody - goat anti-rabbit HRP (ab97051)

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

    Lane 1 : HeLa Cell lysate
    Lane 2 : HeLa Cell lysate
    Lane 3 : 293 Cell lysate
    Lane 4 : 3T3 Mouse Cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    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

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

    Lane 1 : HeLa Cell lysate at 20 ug
    Lane 2 : HeLa Cell lysate at 20 ug
    Lane 3 : 293 Cell lysate at 20 ug
    Lane 4 : 3T3 Mouse Cell lysate at 20 ug

    Secondary
    Rabbit polyclonal to Mouse IgG H&L (HRP) (ab6728) at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size : 42kD

    Exposure time : 10 second

References for Anti-beta Actin antibody [mAbcam 8226] (ab8226)

This product has been referenced in:
  • Nakajima K  et al. Establishment of new predictive markers for distant recurrence of colorectal cancer using lectin microarray analysis. Cancer Med 4:293-302 (2015). IF . Read more (PubMed: 25355679) »
  • Kumari D  et al. Identification of fragile X syndrome specific molecular markers in human fibroblasts: a useful model to test the efficacy of therapeutic drugs. Hum Mutat 35:1485-94 (2014). Human . Read more (PubMed: 25224527) »

See all 197 Publications for this product

Product Wall

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 b...

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Application Western blot
Loading amount 25 µg
Gel Running Conditions Reduced Denaturing (12% polyacrylamide)
Sample Human Cell lysate - whole cell (Huh7.5 hepatoma cell line)
Specification Huh7.5 hepatoma cell line
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Nov 06 2014

Application Immunohistochemistry (Frozen sections)
Sample Human Tissue sections (spleen)
Specification spleen
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 22°C
Fixative Acetone
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Submitted Sep 25 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 22°C
Sample Human Cell (HeLa)
Specification HeLa
Permeabilization No
Fixative Methanol
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Submitted Sep 25 2014

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (4-12%)
Sample Human Cell lysate - whole cell (HepG2)
Specification HepG2
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Sep 25 2014

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (4-12)
Sample Mouse Tissue lysate - whole (liver)
Specification liver
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Sep 25 2014

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (4-12)
Sample Rat Tissue lysate - whole (liver)
Specification liver
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Sep 22 2014

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (10)
Sample Zebrafish Tissue lysate - whole (Larvae 5hpf)
Specification Larvae 5hpf
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
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Submitted Aug 22 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (10)
Sample Rat Cell lysate - whole cell (Primary Neurons)
Specification Primary Neurons
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C
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Submitted Aug 22 2014

Application Western blot
Loading amount 40 µg
Gel Running Conditions Reduced Denaturing
Sample Human Cell lysate - whole cell (hMSCs)
Specification hMSCs
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: rt°C
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Submitted Nov 13 2013

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