Actin c 2

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Actin (C-2) is a monoclonal antibody that specifically recognizes the C-terminus of actin, a ubiquitous structural protein found in eukaryotic cells. This antibody can be used to detect and quantify actin levels in various biological samples.

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Lab products found in correlation

Epr3324, by Abcam (3 mentions) Protease and phosphatase inhibitors, by Santa Cruz Biotechnology (2 mentions) Supersignal west pico, by Thermo Fisher Scientific (2 mentions) Parp1 f 2, by Santa Cruz Biotechnology (2 mentions) Nitrocellulose membrane, by Cytiva (2 mentions) Bca assay, by Thermo Fisher Scientific (2 mentions) Caspase 3, by Cell Signaling Technology (1 mentions) C raf, by Cell Signaling Technology (1 mentions) β actin antibody ac 15, by Merck Group (1 mentions) Odyssey ir imaging system, by LI COR (1 mentions) Bcl xl, by Cell Signaling Technology (1 mentions) Mcl 1, by Cell Signaling Technology (1 mentions) Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions) Protease and phosphatase inhibitor, by Merck Group (1 mentions) Alexa fluor 680 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Phospho fak y397, by Cell Signaling Technology (1 mentions) Irdye 800 conjugated goat anti mouse igg, by Rockland Immunochemicals (1 mentions) Odyssey imaging system, by LI COR (1 mentions) Protease inhibitor, by Promega (1 mentions) P38 mapk 9212, by Cell Signaling Technology (1 mentions)

14 protocols using actin c 2

Immunoblotting of Cell Signaling Proteins

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Immunoblotting was performed with the following antibodies: Bax (D2E11, #5023), Bcl-xL (#2762), Caspase-3 (#9662), Caspase-9 (#9502), p-Cdc2 (Tyr15) (#9111), Cdc2 (POH1, #9116), p-CDK2 (Thr160) (#2561), p-Chk2 (Thr68) (C13C1, #2197), Chk2 (#2662), c-Raf (#9422), CDK2 (78B2, #2546), Cyclin D1 (#2922), Cyclin E1 (D7T3U, #20808), DUSP4 (D9A5, #5149), DUSP6 (#39441), p-Erk1/2 (Thr202/Tyr204) (D13.14.4E, #4370), Erk1/2 (137F5, #4695), Mcl-1 (D5V5L, #39224), MDM2 (D1V2Z, #86934), p-MEK1/2 (Ser217/221) (41G9, #9154), MEK1/2 (47E6, #9126), p21 (12D1, #2947), p-p53 (Ser15) (16G8, #9286), p53 (7F5, #2527), PARP (46D11, #9532), PUMA (D30C10, #12450), p-Rb (Ser789) (#9307), (all from Cell Signaling Technology (Boston, MA, USA)), Actin (C-2, #sc-8432 AF790, Santa Cruz Biotechnology), and β-actin antibody (AC-15, #A5441, Sigma-Aldrich). Immunoblotting signals were visualized using an Odyssey IR imaging system (Li-Cor Biosciences, Lincoln, NE, USA). Image Studio software v4.0 was used for analysis of the bands.

Pairawan S., Akcakanat A., Kopetz S., Tapia C., Zheng X., Chen H., Ha M.J., Rizvi Y., Holla V., Wang J., Evans K.W., Zhao M., Busaidy N., Fang B., Roth J.A., Dumbrava E.I, & Meric-Bernstam F. (2022). Combined MEK/MDM2 inhibition demonstrates antitumor efficacy in TP53 wild-type thyroid and colorectal cancers with MAPK alterations. Scientific Reports, 12, 1248.

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Western Blot Analysis of FAK Signaling

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Total cell lysates were prepared in standard RIPA extraction buffer containing protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, Missouri). Thirty micrograms of protein were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL). The membranes were immunoprobed with FAK, Phospho-FAK (Y397) (Cell Signaling, Danvers, MA) or Actin (C-2) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight separately. Next, membranes were incubated with IRDye 800-conjugated goat anti-mouse IgG (Rockland, Immunochemicals, Gibertsville, PA) or Alexa Fluor 680 goat anti-rabbit IgG (Life Technologies, Grand Island, NY) as secondary antibodies. Bands were detected using Li-Cor Odyssey Imaging System (Li-Cor, Lincoln, NE).

Liu D., Zhou D., Wang B., Knabe W.E, & Meroueh S.O. (2015). A New Class of Orthosteric uPAR•uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR•Vitronectin Interaction. ACS chemical biology, 10(6), 1521-1534.

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Immunoblotting analysis of T-cell signaling

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Treated cell pellets were suspended in lysis buffer with a cocktail of protease inhibitors (Promega, USA). Protein detection by western blot was performed by electrophoretic transfer of equal amounts of SDS-PAGE separated proteins to polyvinyl difluoride (PVDF) membranes (Biorad, USA), incubated with the following primary antibodies: ZAP-70 #2705, LAT #4553, Phosphorylated-LAT #3584, SLP76 #4958 Phospho-SLP76 #92711, p-ZAP70 #2717, p38 MAPK #9212 (Cell Signalling Technology, USA) Actin (C-2; Santa Cruz Biotechnology Inc., USA) and GADPH (6C5; Santa Cruz Biotechnology Inc., USA). Either a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology Inc., USA) or an HRP-conjugated mouse IgG kappa binding protein (m-IgGκ BP) (Santa Cruz Biotechnology Inc., USA) was used for primary antibody detection via chemiluminescence (GE Healthcare, UK).

de Mel S., Mustafa N., Selvarajan V., Azaman M.I., Jaynes P.W., Venguidessane S., Phuong H.M., Alnaseri Z.T., Phyu T., Girard L.P., Chng W.J., Wardyn J., Li Y., An O., Yang H., Ng S.B, & Jeyasekharan A.D. (2022). T and NK cell lymphoma cell lines do not rely on ZAP-70 for survival. PLoS ONE, 17(1), e0261469.

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Quantification of Phosphorylated Sphingosine Kinase 1

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Cell pellets were lysed with ice-cold RIPA lysis buffer supplemented with protease inhibitor (Thermo scientific #A32965) and Phosphatase inhibitor (Thermo scientific #A32957). Protein concentrations of the clarified lysates were determined with the DC Protein Assay (BioRad). 50ug of total protein from each sample were resolved on 10% SDS-PAGE. Gels were then transferred to nitrocellulose membrane and were immunoblotted for proteins of interest (SK1 and p-SK1). Actin was used for loading controls. The following antibodies were used: Actin (C-2) (Santa Cruz Biotechnology sc-8432), Anti-SPHK1 antibody (Abcam ab71700), and SPHK1-Phospho-Ser225 Antibody (Proteintech 19561–1-AP). Proteins of interest were visualized and quantified by the Li-Cor Odyssey Classic or CLx imaging system and the Image Studio software package.

Speirs M.M., Swensen A.C., Chan T.Y., Jones P.M., Holman J.C., Harris M.B., Maschek J.A., Cox J.E., Carson R.H., Hill J.T., Andersen J.L., Prince J.T, & Price J.C. (2019). Imbalanced sphingolipid signaling is maintained as a core proponent of a cancerous phenotype in spite of metabolic pressure and epigenetic drift. Oncotarget, 10(4), 449-479.

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Analyzing Protein Expression in Muscle and Fat

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INS-1 cells, C2C12 myotubes, skeletal muscle and interscapular brown fat tissue were mechanically homogenized in ice-cold lysis buffer and centrifuged at 14,000g for 10 min at 4 °C. The resulting supernatant was separated by SDS–PAGE, and immunoblotted with antibodies to sarcomeric myosin heavy chain (MF20; 1:1,000 dilution; Developmental Studies Hybridoma Bank, University of Iowa), DJ-1 (D-4; 1:500 dilution), UCP1 (M-17; 1:500 dilution), actin (C-2; 1:1,000 dilution) (Santa Cruz Biotechnology), phospho-AMPKα (Thr172) (40H9; 1:1,000 dilution), total AMPKα (23A3; 1:1,000 dilution), phospho-Akt (Ser473) (1:500 dilution), total Akt (1;1,000 dilution), Bcl-xL (H-5; 1:500 dilution), GAPDH (14C10; 1:5,000 dilution) or α/β-tubulin (1:1,000 dilution) (Cell Signalling Technology). Protein band intensity was quantified by ImageJ software.

Shi S.Y., Lu S.Y., Sivasubramaniyam T., Revelo X.S., Cai E.P., Luk C.T., Schroer S.A., Patel P., Kim R.H., Bombardier E., Quadrilatero J., Tupling A.R., Mak T.W., Winer D.A, & Woo M. (2015). DJ-1 links muscle ROS production with metabolic reprogramming and systemic energy homeostasis in mice. Nature Communications, 6, 7415.

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Butyrate and Cytokine Regulation of ICAM-1 and MAPK

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After stimulation with butyrate for 24 h and the inflammatory cytokines IL1β and TNFα for another three hours, HSC-2 cells were extracted with SDS buffer and inhibitors of protease (PhosSTOP with cOmplete; Sigma-Aldrich, St. Louis, MO, USA), divided by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman, GE Healthcare, General Electric Company, Fairfield, CT, USA). Thereafter, membranes were submitted to blocking process for 2 h and exposed to the first antibodies (mouse ICAM-1 G-5 and actin C-2 both at 200 ng/mL; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 24 h. In another series, HSC-2 cells were exposed to butyrate for 24 h and to the cytokines for 30 min before exposed to antibodies against phosphorylated and complete p38 and c-Jun N terminal protein kinase MAPK (both Cell Signaling Technologies, Danvers, MA, USA). Then, proteins were detected by the appropriate HRP-conjugated secondary antibody at 40 ng/mL (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, chemiluminescence detection (Clarity ECL Western Blot Substrate kit, Bio-Rad Laboratories, Hercules, CA, USA) was performed with a ChemiDoc MP System (Bio-Rad Laboratories, Hercules, CA, USA).

Magrin G.L., Di Summa F., Strauss F.J., Panahipour L., Mildner M., Magalhães Benfatti C.A, & Gruber R. (2020). Butyrate Decreases ICAM-1 Expression in Human Oral Squamous Cell Carcinoma Cells. International Journal of Molecular Sciences, 21(5), 1679.

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Protein Extraction and Western Blot

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Cells were lysed in ice-cold RIPA with protease and phosphatase inhibitors (Santa Cruz, Dallas, TX). Whole-cell extracts were prepared by centrifugation (14,000 × g, 15 min) to remove insoluble components. Protein concentrations were determined by BCA assays (Thermo Scientific, Waltham, MA) and loading volumes were normalized. Proteins were then separated by 8% or 4–20% gradient SDS-PAGE (Bio-Rad, Hercules, CA) and transferred to PVDF membranes. Primary antibodies for protein detection included: phospho-H2AX (γH2AX, JBW301, Millipore, Billerica, MA), PARP1 (F-2, Santa Cruz), PAR (Trevigen, Gaithersburg, MD), Actin (C-2, Santa Cruz), XRCC1 (mouse monoclonal, Abcam, Cambridge, UK), small subunit μ-calpain (EPR3324, Abcam). Primary hybridizations were performed in Sigma casein blocking buffer at 4 °C overnight. Secondary HRP-conjugated antibodies were incubated for 1 h at room temperature, followed by detection with SuperSignal West Pico (Thermo Scientific). Bands were quantified by mean intensity using NIH ImageJ, and normalized to actin.

Chakrabarti G., Silvers M.A., Ilcheva M., Liu Y., Moore Z.R., Luo X., Gao J., Anderson G., Liu L., Sarode V., Gerber D.E., Burma S., DeBerardinis R.J., Gerson S.L, & Boothman D.A. (2015). Tumor-selective use of DNA base excision repair inhibition in pancreatic cancer using the NQO1 bioactivatable drug, β-lapachone. Scientific Reports, 5, 17066.

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Western Blot Analysis of Cell Signaling

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Transfected cells were collected and lysed in lysate buffer (Beyotime, Beijing, China). Protein concentration was determined using bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA), and protein samples were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membrane (Whatman, Dassel, Germany). After blocking with 5% nonfat milk for 2 h, the membranes were incubated with the primary antibodies overnight, and horseradish peroxidase-conjugated secondary antibody was used for 2 h. Finally, protein bands were visualized using the enhanced chemiluminescence (ECL) reagent (GE Healthcare, Little Chalfont, UK), and optical density was calculated by ImageJ software version 1.49 (National Institutes of Health, Bethesda, MD, USA).
Antibodies used were as follows: actin (C-2) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p27 (ab54563), p21 (ab80633), Bcl-2 (ab692), Bax (ac32503), cleaved caspase 3 (ab32042), procaspase 3 (ab2171), and horseradish peroxidase-conjugated anti-mouse (ab6785) and anti-rabbit antibodies (ab6721) were purchased from Abcam (Cambridge, MA, USA).

Wang W., Chen J., Dai J., Zhang B., Wang F, & Sun Y. (2016). MicroRNA-16-1 Inhibits Tumor Cell Proliferation and Induces Apoptosis in A549 Non-Small Cell Lung Carcinoma Cells. Oncology Research, 24(5), 345-351.

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Evaluating Cellular Stress Responses

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Cells were lysed in ice-cold RIPA with protease and phosphatase inhibitors (Santa Cruz, Dallas, TX, USA). Whole-cell extracts were prepared by centrifugation at 14 000 × g to remove insoluble components. Protein concentration was determined by BCA assay (Thermo Scientific, Waltham, MA, USA) and loading volume was normalized. Extracts were run on 8 or 4–20% (Bio-Rad, Hercules, CA, USA) gradient acrylamide SDS-PAGE gels and transferred to PVDF membrane. Primary antibodies for protein detection included: phospho-H2A.X (JBW301, Millipore, Billerica, MA, USA), PARP1 (F-2, Santa Cruz), PAR (Trevigen, Gaithersburg, MD, USA), Actin (C-2, Santa Cruz), visfatin/NAMPT (rabbit polyclonal, Abcam, Cambridge, UK), small subunit calpain (EPR3324, Abcam). Primary hybridization was carried out in Sigma casein blocking buffer at 4 ° overnight. Secondary HRP conjugated antibodies were incubated for 1 h at room temperature, followed by detection with SuperSignal West Pico (Thermo Scientific). Bands were quantified by mean intensity in ImageJ and normalized to the actin band intensity to control for loading variation.

Moore Z., Chakrabarti G., Luo X., Ali A., Hu Z., Fattah F.J., Vemireddy R., DeBerardinis R.J., Brekken R.A, & Boothman D.A. (2015). NAMPT inhibition sensitizes pancreatic adenocarcinoma cells to tumor-selective, PAR-independent metabolic catastrophe and cell death induced by β-lapachone. Cell Death & Disease, 6(1), e1599-.

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Western Blot Protein Detection

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Western blots were carried out as previously described [25 (link)]. Primary antibodies for protein detection included: GLS1 (ab93434, Abcam), PARP1 (F-2, Santa Cruz), PAR (Trevigen, Gaithersburg, MD), Actin (C-2, Santa Cruz), small subunit calpain (EPR3324, Abcam), and 53BP1 (Bethyl Laboratories).

Chakrabarti G., Moore Z.R., Luo X., Ilcheva M., Ali A., Padanad M., Zhou Y., Xie Y., Burma S., Scaglioni P.P., Cantley L.C., DeBerardinis R.J., Kimmelman A.C., Lyssiotis C.A, & Boothman D.A. (2015). Targeting glutamine metabolism sensitizes pancreatic cancer to PARP-driven metabolic catastrophe induced by ß-lapachone. Cancer & Metabolism, 3, 12.

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