Mouse anti β actin c4

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Mouse anti-β actin (C4) is a primary antibody that recognizes the β-actin protein, a ubiquitous cytoskeletal protein found in all eukaryotic cells. It is used as a loading control in Western blotting and other protein detection applications.

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Lab products found in correlation

Bca kit, by Thermo Fisher Scientific (6 mentions) Protease inhibitor cocktails and phosphatase inhibitors, by Merck Group (2 mentions) Goat anti rabbit igg, by LI COR (2 mentions) Image studio lite software, by LI COR (2 mentions) Immobilon fl pvdf membrane, by Merck Group (2 mentions) Rabbit anti stat3 79d7, by Cell Signaling Technology (2 mentions) Odyssey infrared imaging system, by LI COR (2 mentions) Mouse anti ha f7, by Santa Cruz Biotechnology (2 mentions) Mouse anti gapdh 6c5, by Santa Cruz Biotechnology (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Alexa fluor 568, by Thermo Fisher Scientific (2 mentions) Mouse anti flag antibody, by Merck Group (1 mentions) Top green qpcr supermix, by Transgene (1 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Celltiter glo luminescent cell viability assay kit, by Promega (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Mg132, by Merck Group (1 mentions) Caspase glo 3 7 assay kit, by Promega (1 mentions) Ab32136, by Abcam (1 mentions)

16 protocols using mouse anti β actin c4

Western Blot Analysis of STAT3 and pSTAT3

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Retina and RPE-choroid were lysed in RIPA buffer with protease inhibitor cocktails and phosphatase inhibitors (Sigma-Aldrich). The protein concentration was determined by BCA kit (Thermo Scientific). Thirty microgram protein was loaded on a 10% SDS-PAGE gel and transferred to an Immobilon-FL PVDF membrane (Millipore, Watford, UK). The membranes were incubated sequentially with rabbit anti-STAT3 (79D7, 1:1000) or rabbit anti-pSTAT3 (Tyr705, D3A7, 1:1000) (both from Cell Signalling, Danvers, MA, USA), followed by goat anti-rabbit IgG (1:10,000, Li-COR Biosciences, Cambridge, UK). The membranes were imaged with Odyssey infrared imaging system (Li-COR Biosciences), and analysed by ImageStudioLite Software (Li-COR Biosciences). The mouse anti-β actin (C4, 1:10,000, Santa Cruz, Dallas, Texas, USA) was used as a housekeeping control protein.

Chen M., Lechner J., Zhao J., Toth L., Hogg R., Silvestri G., Kissenpfennig A., Chakravarthy U, & Xu H. (2016). STAT3 Activation in Circulating Monocytes Contributes to Neovascular Age-Related Macular Degeneration. Current Molecular Medicine, 16(4), 412-423.

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Protein Expression and Quantification

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Mouse anti-HA (F-7), mouse anti-GAPDH (6C5) and mouse anti-β-actin (C4) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The mouse anti-flag antibody and protease inhibitor cocktail were obtained from Sigma (Saint Louis, MO, USA). The MCL1 and ubiquitin antibodies were obtained from Proteintech Company. The CellTiter-Glo Luminescent Cell Viability Assay Kit and Caspase-Glo^® 3/7 Assay Kit were obtained from Promega Company. MG-132 was obtained from Merck Millipore Company. ABT-263 was obtained from Selleck Company. Sorafenib was obtained from MCE Company. The TRIZOL reagent was obtained from Invitrogen. The Top Green qPCR SuperMix was obtained from Transgen Biotech.

Wu L., Lin Y., Feng J., Qi Y., Wang X., Lin Q., Shi W., Zheng E., Wang W., Hou Z., Lin H., Yu C., He Y., Xu Y., Yang H., Lin L, & Li L. (2019). The deubiquitinating enzyme OTUD1 antagonizes BH3-mimetic inhibitor induced cell death through regulating the stability of the MCL1 protein. Cancer Cell International, 19, 222.

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Alzheimer's Disease Pathology Analysis

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Thioflavin-S was obtained from Sigma-Aldrich (T1892). The inhibitor of ubiquitin proteasome system, MG-132 (S, R, S), the inhibitor of Transforming growth factor β (TGF-β) signaling pathway, SB431542 and the inhibitor of mammalian target of rapamycin (mTOR) signaling pathway, rapamycin (Sirolimus) were all purchased from Selleck (Houston, TX, USA). MG-132, SB431542 and rapamycin were all dissolved in Dimethyl sulfoxide (DMSO) and added to the medium 1 h before the treatment of brain extracts. Corresponding with the DMSO content of each treatment, DMSO at a final concentration of 0.5% was added in the control groups. The primary antibodies were: mouse anti-Aβ_1–16 (6E10; SIG-39320, Covance, Princeton, NJ, USA), rabbit anti-APP (Ab32136, Abcam, UK), rabbit anti-APP C-terminal fragments (CTF; A8717, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-β secretase (anti-BACE1; 5606S, Cell Signal, Danvers, MA, USA), mouse anti-β-actin (C4, Santa Cruz, Dallas, TX, USA), rabbit anti-glial fibrillary acidic protein (anti-GFAP; AB5804, Millipore, USA), mouse anti-GFAP (3670S, Cell Signal, Danvers, MA, USA), rabbit anti-ionized calcium binding adapter molecule 1 (anti-Iba1; 019-19741, Wako, Japan), mouse anti-t-Tau (Tau-5, Millipore, USA) and rabbit anti-p-Tau (pSer202, Life-Span BioSciences, San Jose, CA, USA).

Yang Y., Wang M., Yang P., Wang Z., Huang L., Xu J., Wang W., Yu M., Bu L., Fei J, & Huang F. (2018). The Aβ Containing Brain Extracts Having Different Effects in Alzheimer’s Disease Transgenic Caenorhabditis elegans and Mice. Frontiers in Aging Neuroscience, 10, 208.

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Antibodies for Stress Response Signaling

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Rabbit antibodies against Phospho-eIF2α (Ser51), eIF2α, PKR were purchased from Cell Signaling Technology (New England Biolabs, Pickering, ON). Phospho-PKR (pT446), P-PKR (Thr 446), PKR (B-10), PACT, Bcl-2, Bax antibodies were from Epitomics (Burlingame, CA) or Santa Cruz (Santa Cruz, CA). Affinity purified goat polyclonal anti-GFP (EU4) (Eusera, Edmonton, AB) was utilized for immunoprecipitation and mouse monoclonal anti-GFP (B-2) (Santa Cruz) for immunoblotting using GFP-tagged plasmids. Rabbit (A2066, Sigma-Aldrich Canada, Oakville, ON) or mouse anti-β-actin (C4, Santa Cruz), goat anti-FLAG (Santa Cruz) and mouse monoclonal anti-HSP60 (H-1) (Santa Cruz) antibodies were used for loading controls. Secondary antibodies were purchased from GE Healthcare (Baie d’Urfe, Quebec) or Santa Cruz. Tm and IFN-α were from Sigma-Aldrich Canada.

Tang Y., Wang Z., Yang J., Zheng W., Chen D., Wu G., Sandford R., Tang J, & Chen X.Z. (2017). Polycystin-1 inhibits eIF2α phosphorylation and cell apoptosis through a PKR-eIF2α pathway. Scientific Reports, 7, 11493.

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Western Blotting for SNAP25 Cleavage

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Full western blotting methodology is available in supplemental methods. Briefly, to measure onabotA cleavage of SNAP25 in TG neurons cultured from wild-type mice, treated cultured TG neurons were lysed using modified RIPA buffer, sonicated, and centrifuged to be used for western blot analysis. Protein was quantified for equal sample loading (BCA Protein Assay Kit, ThermoFisher Scientific), samples were electrophoresed on polyacrylamide SDS gels (Biorad), and proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. Blots were blocked in buffer for one hour at room temperature (RT) before overnight incubation at 4°C with the following primary antibodies in blocking buffer: mouse anti-β-Actin (C4) (Santa Cruz Biotechnology, sc-47778) and recombinant human anti-SNAP25₁₉₇ (Allergan plc, Ab632), a highly selective antibody for the cleaved epitope of SNAP25 (33 (link)). Blots were washed, incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; Jackson ImmunoResearch), and developed by enhanced chemiluminescence.

Morgenstern J.P., Griffith I.J., Brauer A.W., Rogers B.L., Bond J.F., Chapman M.D, & Kuo M.C. (1991). Amino acid sequence of Fel dI, the major allergen of the domestic cat: protein sequence analysis and cDNA cloning.. Proceedings of the National Academy of Sciences of the United States of America, 88(21), 9690-9694.

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Antibody Characterization and Validation

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Antibodies and reagents used for immunoprecipitation and western blotting include mouse anti‐FLAG M2 agarose affinity gel (Sigma‐Aldrich), mouse anti‐DYKDDDDK (Flag) tag (Cell Signaling Technologies, Danvers, MA), mouse anti‐MYC (purified by National Cell Culture from the ATCC hybridoma CRL‐1729 for MYC 1‐9E10.2), mouse anti‐A.v. monoclonal antibody for green fluorescent protein (JL‐8; Takara Bio, Kusatsu, Japan; recognizes VC), rabbit anti‐GFP (D5.1 Cell Signaling Technology 2956S; recognizes VN), mouse anti‐β‐actin (C4, Santa Cruz Biotechnology), anti‐TREK‐1 (Neuromics, Edina, MN), anti‐POPDC1 (early studies used Santa Cruz, goat #sc‐49889 but now discontinued; Sigma‐Aldrich, rabbit #HP018176), and normal mouse or rabbit IgG (Santa Cruz Biotechnology). The rabbit anti‐AC9 antibody was generated and characterized as described (Baldwin et al, 2019 (link)). Note, POPDC protein runs as multiple bands (48–65 kDa) on western blots with altered sizes/patterns in different tissues due to changes in glycosylation patterns (Schindler et al, 2016a (link)). AC9 generally runs as a single band in Sf9 cells but as multiple bands in mammalian cell lines due to variations in glycosylation and dimerization.

Baldwin T.A., Li Y., Marsden A.N., Rinné S., Garza‐Carbajal A., Schindler R.F., Zhang M., Garcia M.A., Venna V.R., Decher N., Brand T, & Dessauer C.W. (2022). POPDC1 scaffolds a complex of adenylyl cyclase 9 and the potassium channel TREK‐1 in heart. EMBO Reports, 23(12), e55208.

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Western Blot Analysis of STAT3 and pSTAT3

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Immunostaining and Western Blot Antibodies

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Antibodies used for immunostaining: rabbit- anti Olig2 (EMD Millipore AB9610, 1:500), mouse-anti APC [CC1] (EMD Millipore OP80, 1:500), mouse-anti Ki67 (BD Pharmingen 550609, 1:50), donkey anti-rabbit IgG (H + L) secondary- Alexa Fluor 568 (Thermo Fisher Scientific A10042, 1:300) and donkey anti-mouse IgG (H + L) secondary- Alexa Fluor 488 (Thermo Fisher Scientific A21202, 1:300). These antibodies have been documented in a previous porcine study (Ishibashi et al., 2012 (link)).
Antibodies used for western blots: rabbit-anti MBP (Cell signaling 78896, 1:1,000), rabbit-anti PLP1 (Cell Signaling Technology 85971S, 1:1,000), rabbit-anti HMGCS1 (Cell Signaling Technology 42201S, 1:1,000), rabbit-anti GAPDH (Cell Signaling Technology 5174, 1:1,000), mouse anti-β-Actin C4 (Santa Cruz 47778, 1:1,000), mouse anti-MAG (Santa Cruz 166849, 1:1,000), goat anti-MOG (Novus Biologicals NB300–948, 1:1,000), goat anti-rabbit IgG (H+L)-HRP (Thermo Fisher Scientific G21234, 1:5,000), goat anti-mouse IgG (H+L)-HRP (Thermo Fisher Scientific G16072, 1:5,000), donkey anti-goat IgG (H+L)-HRP (Thermo Fisher Scientific A15999, 1:5,000). Gray matter tissue was used as a negative control.

Ahmed S., Travis S.D., Díaz-Bahamonde F.V., Porter D.D., Henry S.N., Mykins J., Ravipati A., Booker A., Ju J., Ding H., Ramesh A.K., Pickrell A.M., Wang M., LaConte S., Howell B.R., Yuan L, & Morton P.D. (2021). Early Influences of Microbiota on White Matter Development in Germ-Free Piglets. Frontiers in Cellular Neuroscience, 15, 807170.

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Western Blot Analysis of HCV Proteins

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Cells that were cultured in six-well plates in the presence or absence of BBR treatment were lysed with RIPA buffer (Sigma) that was supplemented with cOmplete^TM Tablets Mini Protease Inhibitor Cocktail (ROCHE; Basel, Switzerland) for 30 min on ice. The cells were then clarified at 12000 RPM for 30 min, followed by protein quantitation using the Bio-Rad Protein Assay Kit II (Bio-Rad Laboratories; Hercules, CA, USA). Afterward, the whole cell lysates were separated using SDS-PAGE, and then transferred to a PVDF membrane for probing with the following primary antibodies: rabbit anti-LC3 antibody (Thermo Fisher Scientific) at 1:1000; mouse anti-HCV NS5A (Millipore; MAB8694) at 1:250; mouse anti-HCV core (C7-50) (Thermo Fisher Scientific) at 1:400; and, mouse anti-β-actin (C4) (Santa Cruz Biotechnology; Santa Cruz, CA, USA) at 1:1000. The secondary antibodies that included goat anti-rabbit IgG H&L HRP (Abcam) and anti-mouse IgG HRP (Thermo Fisher Scientific) were used at a 1:3000 dilution. The membranes were finally overlaid with ECL (Bio-Rad) before acquiring the images while using the ChemiDoc-ItTS2 imager (UVP; Upland, CA, USA).

Tai C.J., Jassey A., Liu C.H., Tai C.J., Richardson C.D., Wong S.H, & Lin L.T. (2020). Targeting Autophagy Augments Berberine-Mediated Cell Death in Human Hepatoma Cells Harboring Hepatitis C Virus RNA. Cells, 9(4), 908.

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Studying RIG-I and MEX3 Regulation

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The pCMV6-Entry-Myc-DDK-RNF122 (RC210868), pCMV6-Entry-Myc-DDK-MEX3A (RC215359), pCMV6-Entry-Myc-DDK-MEX3C (RC221125), pCMV6-AC-GFP-DDX58 (RG217615), non-effective Scrambled shRNA Cassette in pGFP-C-shLenti Vector (shCTR, TR30021), pGFP-C-shLenti-MEX3A (TL308061B (#1) and TL308061C (#2)) were purchased from Origene (Rockville, MD, USA).
Mouse anti-RIG-I D-12 (sc-376845, 1:1000 for WB, 1:100 for IHC), anti-RIG-I HRP conjugated D-12 (sc-376845, 1:2000), mouse anti-HA-probe F-7 HRP (sc-7392 HRP, 1:1000) and mouse anti-β-Actin C4 (sc-47778, 1:2000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Flag M2 HRP (A8592, 1:1000) was purchased from Sigma-Aldrich. Rabbit anti-RIG-I (D14G6, 1:1000) and Rabbit anti-PARP (95426S) were purchased from Cell Signaling (Beverly, MA, USA). Rabbit anti-MEX3A (ab79046, 1:1000 for WB, 1:100 for IHC) was purchased from Abcam (Cambridge, UK). HRP-conjugated secondary antibodies were purchased from Bethyl Laboratories (Montgomery, TX, USA).
Where indicated, cells were treated with MG132 (50 µM; Calbiochem, Nottingham, UK) for 4 h, Cycloheximide (100 µM, Sigma Aldrich) at indicated time.

Bufalieri F., Caimano M., Lospinoso Severini L., Basili I., Paglia F., Sampirisi L., Loricchio E., Petroni M., Canettieri G., Santoro A., D’Angelo L., Infante P, & Di Marcotullio L. (2020). The RNA-Binding Ubiquitin Ligase MEX3A Affects Glioblastoma Tumorigenesis by Inducing Ubiquitylation and Degradation of RIG-I. Cancers, 12(2), 321.

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