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

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
  • 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.

    (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.

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
    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.

  • 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
    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).

  • 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

  • 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.

  • 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)

    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
  • 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
  • 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

    Lane 1 : HeLa cell lysate
    Lane 2 : HeLa cell lysate
    Lane 3 : HEK293 cell lysate
    Lane 4 : NIH 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
  • 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.

  • 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:
  • Zhang Z  et al. Sepia ink oligopeptide induces apoptosis and growth inhibition in human lung cancer cells. Oncotarget 8:23202-23212 (2017). WB ; Human . Read more (PubMed: 28423568) »
  • Martins CO  et al. Rheumatic Heart Disease and Myxomatous Degeneration: Differences and Similarities of Valve Damage Resulting from Autoimmune Reactions and Matrix Disorganization. PLoS One 12:e0170191 (2017). WB ; Human . Read more (PubMed: 28121998) »

See all 525 Publications for this product

Customer reviews and Q&As

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...

Read More
Application
Western blot
Sample
Rat Tissue lysate - whole (Retina)
Gel Running Conditions
Reduced Denaturing (12% gel)
Loading amount
20 µg
Specification
Retina
Blocking step
Licor Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Aug 01 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Tissue lysate - whole (fat)
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (gel 10%)
Loading amount
20 µg
Specification
fat
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Mar 14 2017

Application
Western blot
Sample
Human Tissue lysate - whole (HEK cells)
Gel Running Conditions
Reduced Denaturing (12% gel)
Loading amount
20 µg
Specification
HEK cells
Blocking step
Licor Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Feb 03 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Zebrafish Tissue lysate - whole (Retina)
Gel Running Conditions
Reduced Denaturing (12% gel)
Loading amount
20 µg
Specification
Retina
Blocking step
Licor Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Feb 03 2017

Application
IHC - Wholemount
Sample
Zebrafish Cell (Whole Body)
Specification
Whole Body
Username

Abcam user community

Verified customer

Submitted Nov 25 2014

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
Username

Abcam user community

Verified customer

Submitted Nov 06 2014

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (5-20)
Sample
Human Cell lysate - whole cell (HepaRG HepG2)
Specification
HepaRG HepG2
Blocking step
Milk as blocking agent for 20 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 4°C
Username

咲織 辻

Verified customer

Submitted Oct 07 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
Username

Abcam user community

Verified customer

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
Username

Abcam user community

Verified customer

Submitted Sep 25 2014

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