Mouse anti β actin a5316

Manufactured by Merck Group

Sourced in United States

Mouse anti-β-actin (A5316) is a monoclonal antibody that specifically binds to the β-actin isoform of the actin protein. Actin is a ubiquitous cytoskeletal protein found in all eukaryotic cells. This antibody is commonly used as a loading control in Western blot analysis to normalize protein expression levels across samples.

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Lab products found in correlation

Ab18181, by Abcam (1 mentions) P0217, by Agilent Technologies (1 mentions) P0447, by Agilent Technologies (1 mentions) Mouse anti flag, by Merck Group (1 mentions) Rabbit anti phospho mtor ser2448, by Cell Signaling Technology (1 mentions) Rabbit anti lc3 pm036, by Medical & Biological Laboratories (1 mentions) Rabbit anti phospho p70 s6 kinase thr389, by Cell Signaling Technology (1 mentions) Ab13248, by Abcam (1 mentions) Ab16080, by Abcam (1 mentions) Goat anti mouse irdye 800, by LI COR (1 mentions) Goat anti rabbit irdye 680, by LI COR (1 mentions) Goat anti mouse alexa 647, by Thermo Fisher Scientific (1 mentions) A11122, by Thermo Fisher Scientific (1 mentions) Mouse anti myc 9b11, by Cell Signaling Technology (1 mentions) Goat anti mouse hrp, by Jackson ImmunoResearch (1 mentions) Prb 101p, by Fortrea (1 mentions) Goat anti rabbit hrp, by Jackson ImmunoResearch (1 mentions) Normal rabbit igg, by Cell Signaling Technology (1 mentions) Rabbit anti cox 4, by Cell Signaling Technology (1 mentions) Anti flag hrp antibody, by Merck Group (1 mentions)

8 protocols using mouse anti β actin a5316

Protein Expression and Immunoblotting in HEK293T Cells

Cited 2 times

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HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal calf serum (FCS; Bodinco BV), 100 units/ml
penicillin, 100 units/ml streptomycin, and 2 mml-glutamine (cell culture medium and supplements were obtained
from Lonza).
Primary antibodies used were mouse anti-HA (ab18181; Abcam), mouse
anti-V5 (37-7500; Invitrogen), mouse anti-β-actin (A5316;
Sigma-Aldrich), mouse anti-FLAG (F3165; Sigma-Aldrich), and rabbit
anti-GFP (30 (link)). As secondary
antibodies, horseradish peroxidase (HRP)-conjugated antibodies were used
(P0447 and P0217; Dako).
The following plasmids were described elsewhere: pASK3 (31 (link)), pcDNA-eGFP (30 (link)), pCMV-FLAG-Ub (32 (link)), pLuc-IFN-β (33 (link)), pEBG-RIG-I_(2CARD) (34 (link)), pcDNA-FLAG-MAVS (35 (link)), and
pEGFP-C1-IRF3_(5D) (36 (link)).

Bailey-Elkin B.A., Knaap R.C., Johnson G.G., Dalebout T.J., Ninaber D.K., van Kasteren P.B., Bredenbeek P.J., Snijder E.J., Kikkert M, & Mark B.L. (2014). Crystal Structure of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Papain-like Protease Bound to Ubiquitin Facilitates Targeted Disruption of Deubiquitinating Activity to Demonstrate Its Role in Innate Immune Suppression. The Journal of Biological Chemistry, 289(50), 34667-34682.

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Antibody Characterization for Protein Analysis

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For Western blotting and immunofluorescence, the following primary antibodies were used: rabbit anti-LC3 [PM036] (1:100 for immunofluorescence; 1:500 for Western blot), rabbit anti-phospho-p62 [PM074] (Ser351) (1:500) from Medical & Biological Laboratories (MBL); rabbit anti-mTOR [#2972] (1:1000), rabbit anti-phospho-mTOR (Ser2448) [#2971] (1:100 for immunofluorescence; 1:1000 for Western blot), rabbit anti-p70S6 kinase [#2708] (1:1000), and rabbit anti-phospho-p70S6 kinase (Thr389) [#2708] (1:1000) from Cell Signaling; guinea pig anti-p62 [P0G-GP62-C] (1:1000) from Progen; rabbit anti-ATP6V0A4 [ab97440] (1:1000) from AbCam; mouse anti-β-tubulin [T4029] (1:1000) and mouse anti-β-actin [A5316] (1:1000) from SIGMA; and rabbit Spin1/SPNS1 (1:100) from original antibodies^{12 (link)}.

Yanagisawa H., Ishii T., Endo K., Kawakami E., Nagao K., Miyashita T., Akiyama K., Watabe K., Komatsu M., Yamamoto D, & Eto Y. (2017). L-leucine and SPNS1 coordinately ameliorate dysfunction of autophagy in mouse and human Niemann-Pick type C disease. Scientific Reports, 7, 15944.

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Antibody Characterization and Validation

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The following antibodies were used: rabbit anti-HA (PRB-101P; Covance), rabbit anti-SPP (ab16080; Abcam), rabbit anti–hepatitis C core (R4210; gift from J. McLauchlan, Medical Research Council–University of Glasgow Centre for Virus Research, Glasgow, Scotland, UK), mouse anti–HO-1 (ab13248; Abcam), mouse anti-myc (9B11; Cell Signaling Technology), mouse anti–β-actin (A5316; Sigma-Aldrich), rabbit anti-RAMP4/4-2 (A18; sc-85114; Santa Cruz Biotechnology, Inc.), mouse anti-calnexin (AF8) and rabbit anti-GFP (A11122; Invitrogen), rabbit anti-US2 (177–5; ER luminal domain; a gift from E. Wiertz, University Medical Center Utrecht, Utrecht, Netherlands), mouse anti–MHC-I (W6/32; ATCC), goat anti–mouse Alexa 647 (Invitrogen), goat anti–mouse IRDye 800 and goat anti–rabbit IRDye 680 (LI-COR Biosciences), and goat anti–mouse HRP and goat anti–rabbit HRP (Jackson ImmunoResearch Laboratories, Inc.).

Boname J.M., Bloor S., Wandel M.P., Nathan J.A., Antrobus R., Dingwell K.S., Thurston T.L., Smith D.L., Smith J.C., Randow F, & Lehner P.J. (2014). Cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins. The Journal of Cell Biology, 205(6), 847-862.

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Antibody Validation for Immunoblotting

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Mouse anti-β-actin (A5316) and anti-Flag-HRP antibodies (A8592) were purchased from Sigma-Aldrich. Anti-Myc-horseradish peroxidase (HRP) antibody (#2040), anti-HA-HRP antibody (#2999), rabbit anti-IRF-3 (#11904), rabbit anti-pIRF-3 (#4947), rabbit anti-Flag (#14793), rabbit anti-LDHA (#3582), rabbit anti-COX IV (#4844), anti-rabbit IgG-HRP-linked antibody (#7074), anti-mouse IgG-HRP-linked antibody (#7076), normal rabbit IgG (#2729) were bought from Cell-Signaling Technologies. Mouse anti-Myc antibody (sc-40), mouse anti-HA antibody (sc-7392), mouse anti-RIG-I (sc-376845), mouse anti-14-3-3ε-horseradish peroxidase (HRP) antibody (sc-23957 HRP), mouse anti-V5 antibody (sc-81594), mouse anti-MAVS (sc-365334), normal mouse IgG (sc-2025) were purchased from Santa Cruz Biotechnology. Mouse anti-HSV ICP27 antibody (P1113) was purchased from Virusys. Anti-γ₁34.5 and NS1 antibodies were described previously [79 (link),80 (link)].

Liu X., Ma Y., Voss K., van Gent M., Chan Y.K., Gack M.U., Gale M J.r, & He B. (2021). The herpesvirus accessory protein γ134.5 facilitates viral replication by disabling mitochondrial translocation of RIG-I. PLoS Pathogens, 17(3), e1009446.

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Comprehensive Western Blot Protein Analysis

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Western blot analysis was performed as previously described (Racca et al., 2011 (link)) and the following antibodies were used: rabbit polyclonal anti-H-Ras (C-20, sc-520, Santa Cruz), mouse monoclonal anti-KLF6 [clone 2c11, previously specified by Gehrau et al. (2011) (link)], rabbit and goat polyclonal anti-p53 (sc6243, Santa Cruz Biotechnology), mouse anti- α -tubulin (T9026, Sigma-Aldrich), mouse anti-β-actin (A5316, Sigma-Aldrich) and donkey-anti-mouse near-Infrared fluorescent secondary antibodies 680CW and 800CW (LI-COR Biosciences, Lincoln, NE, United States). α-tubulin and β-actin were used as loading control. Fluorescence emission was acquired with Odyssey CLx scan (LI-COR, United States). Images are representative of three independent experiments.

Sabatino M.E., Castellaro A., Racca A.C., Carbajosa González S., Pansa M.F., Soria G, & Bocco J.L. (2019). Krüppel-Like Factor 6 Is Required for Oxidative and Oncogene-Induced Cellular Senescence. Frontiers in Cell and Developmental Biology, 7, 297.

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Quantification of Osteocyte Protein Levels

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Whole protein extracts from osteocyte-enriched long bones (cortical bone preparations), calvaria bones, and soft tissue samples were prepared as published.^{(16 ,25 (link))} Protein levels were measured by western blot analysis. Membranes were probed with primary antibodies, rabbit anti-connexin43 (C6219) and mouse anti-β-actin (A5316) (Sigma Aldrich, Saint Louis, MO, USA) diluted 1:1000, overnight at 4°C, followed by incubation with corresponding secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) for 4 hours at room temperature. Membranes were developed with an enhanced chemiluminescence Western blotting substrate kit (Pierce Biotechnology Inc., Rockford, IL, USA). Bands were quantified using ImageJ.

Davis H.M., Aref M.W., Aguilar‐Perez A., Pacheco‐Costa R., Allen K., Valdez S., Herrera C., Atkinson E.G., Mohammad A., Lopez D., Harris M.A., Harris S.E., Allen M., Bellido T, & Plotkin L.I. (2018). Cx43 Overexpression in Osteocytes Prevents Osteocyte Apoptosis and Preserves Cortical Bone Quality in Aging Mice. JBMR Plus, 2(4), 206-216.

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Antibody and Viral Vector Protocol

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The following antibodies were used in this study: mouse anti-Tuj1 (E10045BF; Covance, Princeton, USA), rabbit anti-PTEN (mAb#9559; Cell Signaling Technology, Beverly, USA), chicken anti-GFP (A10262; Invitrogen, Carlsbad, USA), mouse anti-β-actin (A5316; Sigma-Aldrich, St Louis, USA), and mouse anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (AF1186, 1:2000; Beyotime, Shanghai, China) antibodies. The secondary antibodies include Alexa Fluor 488 or 568 and were purchased from Life Technology Molecular Probes, Inc. (Carlsbad, USA). The pAAV-CMV-Cre and pAAV-CMV-GFP virus vectors (titer 1.0×10¹²) were purchased from the HANBIO Company (Shanghai, China). The mouse PTEN small interfering RNA (siRNA) mixture (siPTEN ON-TARGET plus) was purchased from Thermo Scientific Dharmacon (Chicago, USA). The sequences are 5′-UGAUGAUGUAGUAAGGUUU-3′, 5′-GCGCUAUGUAUAUUAUUAU-3′, 5′-GUAGUAGGCUCAAAUAUAC-3′, and 5′-GUUACAAGUUACAUGUUUA-3′. The microRNAs and miR-26a inhibitors were purchased from GenePharma (Suzhou, China). The miR-26a sequence is 5′-UUCAAGUAAUCCAGGAUAGGCU-3′. The sequence of miR-26a inhibitor is complementary to miR-26a, 5′-AGCCUAUCCUGGAUUACUUGAA-3′.

Zhang J., Ma Y.X., Zeng Y.Q., Zhang S.F., Teng Z.Q., Gao J., S, & Liu C.M. (2021). miR-26a promotes axon regeneration in the mammalian central nervous system by suppressing PTEN expression. Acta Biochimica et Biophysica Sinica, 53(6), 758-765.

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Immunoblot Analysis of Signaling Proteins

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The immunoblot analysis was performed as described previously (Lee et al., 2017). The antibodies used for immunoblotting were as follows. Mouse anti‐Flag (F3165) and mouse anti‐β‐actin (A5316) were obtained from Sigma‐Aldrich (St. Louis, MO). Rabbit anti‐p‐Smad1/5/8 was purchased from Cell Signaling Technology (Danvers, MA). Mouse anti‐Smad4 (sc‐7966) and rabbit anti‐Smad1/5/8 (sc‐6031) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Park J.E., Park J.S., Jang S.Y., Park S.H., Kim J., Ki C, & Kim D. (2019). A novel SMAD6 variant in a patient with severely calcified bicuspid aortic valve and thoracic aortic aneurysm. Molecular Genetics & Genomic Medicine, 7(5), e620.

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