Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224)

Overview

  • Product nameAnti-beta Actin antibody [mAbcam 8224] - Loading Control
    See all beta Actin primary antibodies
  • Description
    Mouse monoclonal [mAbcam 8224] to beta Actin - Loading Control
  • SpecificityRecognises a single band at 42kD representing beta Actin.
  • Tested applicationsFlow Cyt, IHC-P, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Chicken, Cow, Cat, Dog, Human, Pig, Xenopus laevis, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, Chinese Hamster, Other
  • Immunogen

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

    (Peptide available as ab13772.)

  • Positive control
    • This antibody gave a positive signal in Human Skeletal Muscle as well as the following whole cell lysates: A431; HEK293; NIH3T3; PC12. In Flow Cytometry, this antibody gave a positive signal in methanol fixed/Tween permeabilised HeLa cells.
  • General notes

     

    This clone works well as a loading control for Xenopus, Drosophila and S.pombe. We recommend using ab8224 instead of ab8226 for these species.

    Note: although S. cerevisae is not known to express beta Actin, Abcam believes a band at similar MW to beta Actin detected in this species corresponds to Actin 1 (Swissprot ID: P60010, based on sequence similarity) - see WB image.

Properties

Applications

Our Abpromise guarantee covers the use of ab8224 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
Flow Cyt Use 1µg for 106 cells.
IHC-P 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 42 kDa (predicted molecular weight: 42 kDa).Can be blocked with Human beta Actin peptide (ab13772). This antibody has been designed for use as a loading control and is ideal for this purpose. Block membrane for 1 hr in 5%BSA. Incubate antibody in TBST for one hour or more.
ICC/IF Use a concentration of 1 µg/ml.

Target

Anti-beta Actin antibody [mAbcam 8224] - Loading Control images

  • IHC image of beta actin staining in human colon formalin fixed paraffin embedded tissue section*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab8224, 3µg/ml overnight at +4°C. An HRP-conjugated secondary (ab6789, 1/2000 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • All lanes : Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) at 1 µg/ml

    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
    Lane 5 : Skeletal Muscle (Human) Tissue Lysate - adult normal tissue

    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 ab8224 overnight at 4°C. Antibody binding was detected using 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 8224] - Loading Control (ab8224) 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

    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 ab8224 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution ab133406

  • All lanes : Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) at 1 µg/ml

    Lane 1 : Drosophila lysate
    Lane 2 : S. pombe lysate
    Lane 3 : S. cerevisiae lysate (Actin 1 - please see note)

    Lysates/proteins at 20 µg per lane.

    Secondary
    Rabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/5000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

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

    Note: although S. cerevisae is not known to express beta Actin, Abcam believes that the band on lane 3 corresponds to Actin 1 (Swissprot ID: P60010, based on sequence similarity).

  • Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) + Xenopus embryo lysate at 20 µg

    Secondary
    Rabbit Anti-Mouse IgG H&L (HRP) (ab6728)
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 42 kDa

    ab8224 used on Xenopus embryo lysate (20 ug of lysate/lane).

    Secondary
    Rabbit polyclonal antibody to Mouse IgG H&L (HRP) (ab6728) developed using the ECL technique.
    Performed under reducing conditions.

    Predicted band size : 42kD

  • All lanes : Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224) at 1/1000 dilution

    Lane 1 : Fruit fly (Drosophila melanogastor) whole cell lysate - Female
    Lane 2 : Fruit fly (Drosophila melanogastor) whole cell lysate - Male

    Lysates/proteins at 100 µg per lane.

    Secondary
    An HRP-conjugated Sheep polyclonal to mouse IgG at 1/10000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 42 kDa


    Exposure time : 2 minutes

    This image is courtesy of an anonymous Abreview

    Blocking step: 5% Milk for 1 hour at 20°C.

    See Abreview

  • Immunohistochemistical detection of beta Actin using antibody [mAbcam 8224] - Loading Control on formaldehyde-fixed paraffin-embedded rat cerebellum sections. Antigen retrieval step: heat mediated Citric acid pH6 buffer.  Permeabilization: No. Blocking step: 1% BSA for 10 mins @ rt°C. Primary antibody dilution 1/1000 for 2 hours in TBS/BSA/azide. Secondary Antibody: anti Mouse Igs conjugated to biotin (1/200). beta Actin appears to be particularly enriched not only in the glomeruli of the Granule cell layer (indicated by red arrowheads ) but also in Microglia (indicated by green arrowheads); All positive microglia appear to be ramified thus not presumed to be activated.

    See Abreview

  • ICC/IF image of ab8224 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab8224, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Overlay histogram showing HeLa cells stained with ab8224 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab8224, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was Mouse IgG3 [MG3-35] (ab18394, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Immunofluorescence analysis of Human Panc-1 cells, staining beta Actin with ab8224.

    Cells were fixed with formaldehyde and blocked with 1% donkey serum in PBST for 1 hour at 22°C. Samples were incubated with primary antibody (1/75 in 1% donkey serum in PBST) for 1 hour at 22°C. A DyLight®488-conjugated donkey anti-mouse polyclonal IgG (1/200) was used as the secondary antibody.

    See Abreview

References for Anti-beta Actin antibody [mAbcam 8224] - Loading Control (ab8224)

This product has been referenced in:
  • Mukherjee S  et al. Potential theranostics application of bio-synthesized silver nanoparticles (4-in-1 system). Theranostics 4:316-35 (2014). WB . Read more (PubMed: 24505239) »
  • Steinhauf D  et al. Silencing motifs in the Clr2 protein from fission yeast, Schizosaccharomyces pombe. PLoS One 9:e86948 (2014). WB ; Schizosaccharomyces pombe . Read more (PubMed: 24475199) »

See all 51 Publications for this product

Product Wall

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

Abcam user community

Verified customer

Submitted Jul 21 2014

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (12% gel)
Sample Mouse Tissue lysate - whole (Skeletal muscle (AT))
Specification Skeletal muscle (AT)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Feb 13 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Mouse Tissue lysate - whole (Mouse whole brain tissue lysate, striatum and cort)
Loading amount 15 µg
Specification Mouse whole brain tissue lysate, striatum and cort
Gel Running Conditions Non-reduced Non-Denaturing (Native) (4-12% Bis-Tris Gel, MES running buffer)
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Mar 11 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (Panc-1)
Specification Panc-1
Fixative Formaldehyde
Permeabilization No
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C
Username

Mr. Armen Petrosyan

Verified customer

Submitted Dec 05 2012

Thank you for your reply and for providing that additional information. This will be very helpful for us to understand what may have contributed to the problems you have encountered.

As discussed, I have now arranged for a different vial of ab...

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Thank you for contacting us today and sorry for the delay in getting back to you.

I am sorry to hear that you have been experiencing problems with the anti-beta Actin antibody (ab8224). As you have used this antibody before with no problems, a...

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Thank you for contacting us yesterday in regards to the Anti-beta Actin antibody [mAbcam8226] (HRP) - Loading Control (ab20272).

I have looked into this case a little further. As we discussed over the phone, the reactivity with Saccharomyces ...

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Thank you for your call today and for your questions.

Since ab9485 and ab8224have not been tested inC.albicans samples,I can offer a discount off a future purchase if you buyone of these antibodiesnow, test it inC.albicans, and submit feedba...

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Thank you for your reply.


The signal does appear to be a bit cleaner with the 5% BSA blocking, but there are still quite a few bands.


I would be happy to send you a free of charge replacement vial, however we only have t...

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Thank you for your reply and for confirming the details of your experiment.


I see that you're using whole cell lysates, but from what species?


Would you be able to try blocking with 5% BSA instead of milk? We do not reco...

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