Overview

  • Product name
  • Description
    Rabbit polyclonal to beta Actin
  • Specificity
    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: IHC-Fr, IP, WB, ICC, Flow Cyt, IHC-FrFl, IHC-P, IHC-P, ICC/IF, ELISAmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Chicken, Guinea pig, Cow, Dog, Human, Pig, Xenopus laevis, Drosophila melanogaster, Fish, Monkey, Zebrafish, Rhesus monkey, Chinese hamster
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human beta Actin aa 1-100. The exact sequence is proprietary.
    (Peptide available as ab28691, ab13772)

  • Positive control
    • WB: Mouse brain tissue lysate and HeLa, A431, HEK293, NIH3T3 and PC12 whole cell lysates. IHC-P: Normal human colon and normal human liver tissues. ICC/IF: NIH3T3 and SV40LT-SMC cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab8227 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
IHC-Fr Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
WB 1/1000 - 1/5000.
ICC Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab171870 - Rabbit polyclonal IgG, 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 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IHC-P Use a concentration of 0.2 µg/ml.
ICC/IF Use a concentration of 1 µg/ml.
ELISA 1/1000.

Target

Images

  • All lanes : Anti-beta Actin antibody (ab8227) 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-Rabbit IgG H&L (Alexa Fluor® 790) (ab175781) at 1/10000 dilution

    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 ab8227 overnight at 4°C. Antibody binding was detected using ab175781 at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

  • ab8227 staining beta Actin in SV40LT-SMC cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab8227 at 1μg/ml (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • IHC image of ab8227 staining beta Actin in rat small intestine formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with EDTA (epitope retrieval solution 2) for 20 mins. The section was then incubated with ab8227, 0.2μg/ml, 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.

  • All lanes : Anti-beta Actin antibody (ab8227) at 0.1 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 : Liver (Rat) Tissue Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

    Performed under reducing conditions.

    Exposure time : 1 minute

    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 ab8227 overnight at 4°C. Antibody binding was detected using an anti-rabbit HRP secondary antibody, and visualised using ECL development solution ab133406

  • ab8227 staining beta Actin in NIH3T3 cells. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab8227 at 1μg/ml (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • IHC image of beta actin staining in a section of formalin-fixed paraffin-embedded normal human colon*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was then incubated with ab8227, 1/1000 dilution, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody (ab6720, 1/1000 dilution) was used to detect the primary, and visualized using an HRP conjugated ABC system. Streptavidin HRP was used, ab7403 at a 1/10000 dilution. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was then counterstained with haematoxylin and mounted with DPX.

    The inset negative control image is taken from an identical assay without primary antibody.

    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

  • All lanes : Anti-beta Actin antibody (ab8227) at 1/1000 dilution

    Lane 1 : HeLa nuclear lysate
    Lane 2 : HeLa whole cell lysate
    Lane 3 : A431 cell lysate
    Lane 4 : Jurkat cell lysate
    Lane 5 : HEK 293 cell lysate
    Lane 6 : HeLa nuclear lysate with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 7 : HeLa whole cell lysate with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 8 : A431 cell lysate with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 9 : Jurkate cell lysate with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 10 : HEK 293 cell lysate with Human beta Actin peptide (ab13772) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

    Exposure time : 5 seconds

    All lanes : Anti-beta Actin antibody (ab8227) at 1/1000 dilution

    Lane 1 : HeLa nuclear lysate at 20 µg/ml
    Lane 2 : HeLa whole cell lysate at 20 µg/ml
    Lane 3 : A431 cell lysate at 20 µg/ml
    Lane 4 : Jurkat cell lysate at 20 µg/ml
    Lane 5 : HEK 293 cell lysate at 20 µg/ml
    Lane 6 : HeLa nuclear lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 7 : HeLa whole cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 8 : A431 cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 9 : Jurkate cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 10 : HEK 293 cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

  • IHC image of beta Actin staining in normal human colon, formalin-fixed and paraffin-embedded tissue*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab8227, 3µg/ml overnight at +4°C. A anti-rabbit HRP secondary antibody (Ab97200, 1/200 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

    The inset negative control image is taken from an identical assay without primary antibody.

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

  • All lanes : Anti-beta Actin antibody (ab8227) at 1/1000 dilution

    Lane 1 : HeLa nuclear lysate at 20 µg/ml
    Lane 2 : HeLa whole cell lysate at 20 µg/ml
    Lane 3 : A431 cell lysate at 20 µg/ml
    Lane 4 : Jurkat cell lysate at 20 µg/ml
    Lane 5 : HEK 293 cell lysate at 20 µg/ml
    Lane 6 : HeLa nuclear lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 7 : HeLa whole cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 8 : A431 cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 9 : Jurkate cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml
    Lane 10 : HEK 293 cell lysate at 20 µg/ml with Human beta Actin peptide (ab13772) at 1 µg/ml

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

  • All lanes : Anti-beta Actin antibody (ab8227) at 1/1000 dilution

    Lane 1 : HeLa cells (Human) at 20 µg
    Lane 2 : 3T3 cells (Mouse) at 20 µg
    Lane 3 : Fish Liver at 20 µg
    Lane 4 : Rabbit Liver at 20 µg
    Lane 5 : MDCK cells (Dog) at 20 µg/ml
    Lane 6 : EBTr cells (Cow) at 20 µg
    Lane 7 : SL-29 cells (Chicken) at 20 µg/ml
    Lane 8 : CHO cells (Chinese Hamster) at 20 µg
    Lane 9 : Xenopus embryo at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

    Exposure time : 30 seconds

    Western blot using ab8227 at 1/1000.

    Lysates:
    Lane 1: HeLa cells (Human)
    Lane 2: 3T3 cells (Mouse)
    Lane 3: Fish Liver
    Lane 4: Rabbit Liver
    Lane 5: MDCK cells (Dog)
    Lane 6: EBTr cells (Cow)
    Lane 7: SL-29 cells (Chicken)
    Lane 8: CHO cells (Chinese Hamster)
    Lane 9: Xenopus embryo

    Secondary ab: anti-rabbit HRP (ab6721)
    Exposure time: 30 sec
    Lysates at 20µg/lane.
    Expected molecular weight: 41.7kDa.

References

This product has been referenced in:
  • Sun Y  et al. Therapeutic effect of apocynin through antioxidant activity and suppression of apoptosis and inflammation after spinal cord injury. Exp Ther Med 13:952-960 (2017). WB ; Rat . Read more (PubMed: 28450925) »
  • Kostarnoy AV  et al. Receptor Mincle promotes skin allergies and is capable of recognizing cholesterol sulfate. Proc Natl Acad Sci U S A 114:E2758-E2765 (2017). WB . Read more (PubMed: 28292894) »

See all 786 Publications for this product

Customer reviews and Q&As

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
Username

Mr. Pedro Guedes Dias

Verified customer

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
Username

Abcam user community

Verified customer

Submitted Aug 22 2014

Application
Immunohistochemistry (Frozen sections)
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Sample
Mouse Tissue sections (Skin)
Specification
Skin
Permeabilization
Yes - 0,3% TritonX
Fixative
Perfusion fixed with paraformaldehyde and picric acid
Username

Mr. Frank Rommel

Verified customer

Submitted Aug 08 2014

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Reduced Denaturing (Biorad Criterion TGX Stain Free Gel, Any kD, Cat. number 5678123)
Sample
Fruit fly (Drosophila melanogaster) Tissue lysate - whole (Drosophila heads)
Specification
Drosophila heads
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted May 02 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (12%)
Sample
Rat Tissue lysate - whole (ovaries)
Specification
ovaries
Blocking step
Milk as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C
Username

Mrs. Henda Nabli

Verified customer

Submitted Oct 01 2013

We do not know if ab8227 stains nuclear actin. It is raised against a sequence from cytoplasmic actin, and we have not tested for cross-reactivity with nuclear actin.

http://www.uniprot.org/uniprot/P60709

I just received an update form lab regarding the epitope for ab79667 and ab113279. Unfortunately the epitope has not been determined so the information is not available. I am sorry we will not be able to suggest these to be paired with ab8227 in sELISA.

I have had the opportunity to discuss our QC methods regarding this product with the lab. Ab17721 is batch tested by us in WB in HeLa, Jurkat and NIH3T3 lysates and also in Immunofluorescence in HeLa cells. Each new AP purified must pass both of these ...

Read More
Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Bat Cell lysate - whole cell (kidney)
Gel Running Conditions
Reduced Denaturing (10% SDS-PAGE)
Loading amount
20000 cells
Specification
kidney
Blocking step
BSA as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Username

Noreen Rapin

Verified customer

Submitted May 24 2017

Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293T)
Gel Running Conditions
Reduced Denaturing (4-10%)
Loading amount
20 µg
Specification
HEK293T
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Nov 08 2016

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