pan Actin Antibody (5J11)

Western Blot: Actin Antibody (5J11) [NBP2-25142] - NBP2-25142 was used to detect GST fusion proteins expressing each of the six human actin proteins. GST-fusion proteins encoding all six full length human actin gene products were run out on SDS-PAGE, transferred to nitrocellulose and reacted with NBP2-25142. The antibody stained all six proteins indicating that it is a panspecific actin antibody.

Immunocytochemistry/Immunofluorescence: Actin Antibody (5J11) [NBP2-25142] - Beta actin was detected in NIH-3T3 cells fixed with methanol using mouse anti-mouse beta-Actin monoclonal antibody (NBP2-25142) at 1:2700 dilution. Cells were stained using Northern Lights 557 conjugated anti-mouse secondary antibody (NL007) and counterstained with DAPI.

Flow Cytometry: Actin Antibody (5J11) [NBP2-25142] - analysis of HeLa cells using mouse Monoclonal Actin antibody (Orange) and Isotype control Antibody (Blue).

Western Blot: Actin Antibody (5J11) [NBP2-25142] - Analysis of HeLa, A431, K562, MCF-7, NIH-3T3, C6, and Rat-2 cell lines.

Western Blot: Actin Antibody (5J11) [NBP2-25142] - lysates of HeLa human cervical epithelial carcinoma cell line, A431 human epithelial carcinoma cell line, and K562 human chronic myelogenous leukemia cell line. PVDF membrane was probed with 0.2 ug/mL mouse anti-Actin monoclonal (NBP2-25142, Novus Biologicals), followed by 1:2000 dilution of HRP- conjugated secondary antibody, donkey anti-mouse IgG (HAF018, R&D Systems).

Western Blot: Actin Antibody (5J11) [NBP2-25142] - Analysis of tissue and cell lysates probed with mouse mAb against actin, NBP2-25142, in red. [1] protein standard, [2] rat brain, [3] mouse brain, [4] NIH-3T3, [5] HEK293, [6] HeLa, [7] SH-SY5Y cells. The same blot was simultaneously probed with chicken pAb to UCHL-1, a marker of neuronal lineage cells, in green.

Immunocytochemistry/Immunofluorescence: Actin Antibody (5J11) [NBP2-25142] - HeLa cells stained with NBP2-25142 (green) and also with the chicken polyclonal antibody to Vimentin (NB300-223, red). The Actin antibody stains the submembranous Actin rich cytoskeleton and also stress fibers, bundles of Actin associated with adhesion sites. The Vimentin antibody stains a quite different cytoskeletal network, the intermediate filaments. The blue stain reveals DNA in the nuclei of these cells.

• Reactivity: Hu, Mu, Rt, Po, Bv, Eq
• Applications: WB, Flow, ICC/IF, IHC
• Clone: 5J11
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Concentration: 1 mg/ml

pan Actin Antibody (5J11) Summary

• Immunogen: An Actin preparation derived from bovine brain.
• Localization: Cytoplasm > cytoskeleton.
• Specificity: This antibody recognizes all six isotypes of Actin.
• Isotype: IgG1
• Clonality: Monoclonal
• Host: Mouse
• Gene: ACTA1
• Purity: Immunogen affinity purified

Applications/Dilutions

• Dilutions:
  • Western Blot 1:1000
  • Flow Cytometry 1:10-1:1000
  • Immunocytochemistry/Immunofluorescence 1:500-1:1000
  • Immunohistochemistry 1:500-1:1000
• Application Notes: This Actin (5J11) antibody is useful for Immunocytochemistry/Immunofluorescence, Immunohistochemistry, and Western Blot, where a band can be seen at ~42 kDa.
• Theoretical MW: 42 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: 50% PBS, 50% Glycerol
• Preservative: 5mM Sodium Azide
• Concentration: 1 mg/ml
• Purity: Immunogen affinity purified

Alternate Names for pan Actin Antibody (5J11)

• ACTA
• actin, alpha 1, skeletal muscle
• alpha skeletal muscle actin
• alpha skeletal muscle
• alpha-actin-1
• ASMA
• CFTD
• CFTDM
• MPFD
• NEM1
• NEM2
• NEM3

Background

Actin is one of the most abundant and highly conserved proteins of eukaryotes. Mammalian Actins are the product of six different genes with differing distribution patterns in cell types and in tissues. The molecular weight of all six proteins is 42 kDa, and one or more actins are found in essentially every type of crude cellular and tissue extract. As a result, antibodies to Actin are widely used. As western blotting standards, they can be used to verify that the various steps of the western blotting procedure have been performed correctly. In addition, Actin is regarded as a "housekeeping" protein which is generally not altered much in expression as a result of experimental manipulations. Thus, quantitation of the Actin band on the western is used as a standard against the band density of other proteins. The Actin isotypes were originally classified as alpha, beta and gamma forms based on their charges which resulted in different mobilities in the isoelectric focusing dimension as seen on 2-dimensional SDS-PAGE. Subsequently, the alpha spot was found to potentially contain three Actin gene products, alpha-skeletal Actin, alpha-vascular smooth muscle Actin, and alpha-cardiac muscle. The beta spot contained a single protein called simply beta Actin, while the gamma spot may contain both gamma-1 Actin and gamma-2, which are enteric and smooth muscle Actins. The six mammalian gene products are very similar as shown by a sequence alignment of the human proteins. The most closely related actin isotypes are 97% identical, while the most distantly related are 94% identical, with most of the variability at the N-terminus. With this level of identity, antibodies to any one protein may recognize all six isotypes, as is the case with clone 5J11. This antibody binds all six isotypes very strongly on western blots, and it is a very effective blotting standard which can work on any cell type or tissue extract. It also works in immunocytochemical experiments, binding strongly and cleanly to filopodia, membrane ruffles and stress fibers, all known to be rich in Actin.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Reviews for pan Actin Antibody (NBP2-25142) (2)

Average Rating: 5

(Based on 2 reviews)

We have 2 reviews tested in 1 species: Human.

reviewed by: Alissa Taylor WB Human 09/24/2014

Summary

• Application: Western Blot
• Sample Tested: Hela whole cell lysate
• Species: Human

reviewed by: Anonymous WB Human 06/17/2014

Summary

• Application: Western Blot
• Sample Tested: 293T and HepG2 lysates
• Species: Human

Blocking

• Blocking Details: 5% BSA in PBS

Primary Anitbody

• Dilution Ratio: 1:2000 diluted in PBS with triton X-100, blot was incubated at room temperature on a rocker

Secondary Antibody

• Secondary Description: anti-mouse IgG HRP conjugate (Jackson Labs)
• Secondary Manufacturer Cat#: 115-005-174
• Secondary Concentration: 1:15000

Details

• Detection Notes: Probed using Visualizer Western Blot Detection Kit (Millipore) and captured on X-ray films, 2 sec exposure

Bioinformatics

• Gene Symbol: ACTA1