beta Actin antibody (AA 2-16)

Catalog No. ABIN1742508

This anti-beta Actin antibody is a Rabbit Polyclonal antibody detecting beta Actin in WB, IHC and ICC. Suitable for Mouse, Rat, Chicken and Zebrafish (Danio rerio). Independently validated for use in Western Blotting. This Primary Antibody has been cited in 3+ publications.

Quick Overview for beta Actin antibody (AA 2-16) (ABIN1742508)

Target: beta Actin (ACTB) (Actin, beta (ACTB))

Reactivity: Mouse, Rat, Chicken, Zebrafish (Danio rerio)

Host: Rabbit

Clonality: Polyclonal

Conjugate: This beta Actin antibody is un-conjugated

Application: Western Blotting (WB), Immunohistochemistry (IHC), Immunocytochemistry (ICC)

Product Details anti-beta Actin Antibody

Binding Specificity: AA 2-16

Cross-Reactivity (Details): May cross-react to alpha- and gamma-actin due to sequence homology.

Purification: Affinity purified with the immunogen. Rabbit serum albumin was added for stabilization.

Immunogen: Synthetic peptide (aa 2-16 of mouse beta-actin) coupled to KLH via a C-terminally added cysteine.

Application Details

Application Notes: WB: 1 : 1000 up to 1 : 5000
IP: not tested yet
ICC: 1 : 200 up to 1 : 500
IHC: 1 : 200

Comment:

ICC: Methanol fixation is recommended.

Restrictions: For Research Use only

Independent Validation (WB)

Validation #029836 (Western Blotting)

by: Kinexus Bioinformatics Corporation

No.: #029836

Date: 06/22/2015

Lot Number: 251003/3

Method validated: Western Blotting

Positive Control: mouse brain, mouse kidney, mouse muscle, mouse spleen, mouse testes

Negative Control: none [ubiquitously expressed]

Notes: A band was observed in the positive controls at the expected size (~42 kDa)

Validation Images

Figure 1. Western blot for beta actin (ACTB). Grey arrowhead indicates the expected molecular weight of ~42 kDa.

Figure 2. Ponceau stain loading control.

Figure 3. Western blot for beta actin (ACTB) using attempted siRNA silencing. Arrowhead indicates the expected molecular weight of ~42 kDa. HeLa cells were transfected with beta actin siRNA (Catalog number 12935141, Life Technologies) using Lipofectamine 2000, according to manufacturer's instructions. Total cell lysates were made from both floating and adherent cells harvested 48 hours and 72 hours after transfection. siRNA silencing of beta actin was not detected.

Full Methods

Primary Antibody:

• Antigen: Beta-Actin, (ACTB) (AA 2-16)
• Catalog number: ABIN1742553
• Lot number: 251003/3
• Dilution: 1:1000

Secondary Antibody:

• Antibody: Donkey anti-Rabbit IgG Antibody (HRP)
• Lot number: F0613
• Dilution: 1:10,000

Full Protocol:

• 1. Cell/tissue total protein lysates were boiled in 1X SDS Sample Buffer containing 1% SDS and 1.25% β-mercaptoethanol at 95°C for 5 minutes prior to loading.
• 2. 15 μg of boiled lysate were loaded and resolved on a 12% SDS-polyacrylamide gel.
• 3. The Precision Plus Protein™ All Blue Prestained Standards from BioRad (161-0373) were used as molecular mass markers.
• 4. Proteins were transferred onto nitrocellulose membrane by tank transfer and protein transfer was confirmed with Ponceau S staining.
• 5. The immunoblot membrane was blocked in 2.5% skim milk and 1.5% BSA solution in TTBS at room temperature for 60 minutes.
• 6. The membrane was washed in TTBS twice for 5 minutes each.
• 7. The membrane was immersed with the protein side up in the antibody solution in TBS and incubated overnight at 4°C with gentle agitation.
• 8. The membrane was rinsed twice with TTBS.
• 9. The membrane was washed in TTBS twice for 5 minutes each.
• 10. The membrane was washed in TTBS once for 15 minutes.
• 11. The membrane was incubated in the HRP-conjugated secondary antibody solution in TBS for 60 minutes at room temperature with gentle agitation.
• 12. The membrane was rinsed twice with TTBS.
• 13. The membrane was washed in TTBS twice for 5 minutes each.
• 14. The membrane was washed in TTBS once for 15 minutes.
• 15. Signals were detected by chemiluminescence (ECL). The blot was scanned for 160 seconds.
• 16. The membrane was rinsed three times with TTBS.
• 17. Incubated in Acidic Glycine Stripping Buffer at room temperature with gentle agitation for 3 times, 10 minutes each.
• 18. The membrane was washed in TTBS 3 times for 5 minutes each.
• 19. Repeated Steps 4-14 with the loading control antibody (for β3−Tubulin).
• 20. Repeated Steps 4-14 with the loading control antibody and its matching secondary antibody. The blot was scanned for 160 seconds.

Experimental Notes: A protein band was observed at the expected target size of β−Actin of 42 kDa in all mouse extracts tested. Lower molecular weight bands of approximately 28-30 kDa were also observed in the mouse kidney and spleen tissue samples. Three main groups of actin have been identified. The β-actins and γ-actins co-exist in most cell types as components of the cytoskeleton, whereas the α-actin are found in muscle tissues. In our experience, we find that the level of actin does vary widely between different cell and tissue types.

Handling

Format: Lyophilized

Reconstitution: For reconstitution add 100 µL H2O to get a 1mg/ml solution of antibody in PBS. Then aliquot and store at -20 °C until use.

Buffer: PBS

Handling Advice: Affinity purified antibodies are less robust than antisera, since protease inhibitors are also removed during purification. Hence, storage at 4 °C for prolonged periods (i.e. several weeks), is not recommended.

Storage: -20 °C

Storage Comment: Unlabeled lyophilized antibodies are stable in this form without loss of quality at ambient temperatures for several weeks or even months. They can be stored at 4°C for several years. Lyophilized antibodies must not be stored in the freezer, they may be destroyed!

References for anti-beta Actin antibody (ABIN1742508)

Lu, Ma, Zhuang, Zhang, Cheng, Shi: "Retinoic acid‑incorporated glycol chitosan nanoparticles inhibit the expression of Ezh2 in U118 and U138 human glioma cells." in: Molecular medicine reports, Vol. 12, Issue 5, pp. 6642-8, (2016) (PubMed).

Baron, Blex, Rohrbeck, Rachakonda, Birnbaumer, Ahnert-Hilger, Brunk: "The α-subunit of the trimeric GTPase Go2 regulates axonal growth." in: Journal of neurochemistry, Vol. 124, Issue 6, pp. 782-94, (2013) (PubMed).

Whitemarsh, Strathman, Chase, Stankewicz, Tepp, Johnson, Pellett: "Novel application of human neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection." in: Toxicological sciences : an official journal of the Society of Toxicology, Vol. 126, Issue 2, pp. 426-35, (2012) (PubMed).

Target Details for beta Actin

Target: beta Actin (ACTB) (Actin, beta (ACTB))

Alternative Name: beta-Actin

Pathways: Myometrial Relaxation and Contraction, Cell-Cell Junction Organization, Maintenance of Protein Location, Phototransduction