Sc 55529

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Sc-55529 is a laboratory equipment product from Santa Cruz Biotechnology. It is designed for use in scientific research and analysis. The core function of this product is to facilitate specific experimental procedures, but a detailed description cannot be provided without the risk of bias or extrapolation.

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Lab products found in correlation

Irdye 800cw goat anti rabbit igg, by LI COR (2 mentions) Irdye 680lt goat anti mouse igg, by LI COR (2 mentions) Odyssey infrared imaging system, by LI COR (2 mentions) Sc 853, by Santa Cruz Biotechnology (2 mentions) β tubulin, by Santa Cruz Biotechnology (2 mentions) Chemidoc xrs imaging system, by Bio-Rad (1 mentions) Image lab 6, by Bio-Rad (1 mentions) M iggκ bp, by Santa Cruz Biotechnology (1 mentions) Sc 7271, by Santa Cruz Biotechnology (1 mentions) Ab7971, by Abcam (1 mentions) Hrp conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions) Sc 516102, by Santa Cruz Biotechnology (1 mentions) Sc 2004, by Santa Cruz Biotechnology (1 mentions) Dmi6000 inverted microscope, by Leica (1 mentions) Rat tail collagen type 1, by Corning (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions) Xcell 2 blot module, by Thermo Fisher Scientific (1 mentions) Sonic dismembrator, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Immobilon p pvdf transfer membrane, by Merck Group (1 mentions)

Spelling variants of this product

Anti-β-tubulin (sc-55529) (3 citations) (β-) tubulin (sc-55529) (2 citations)

10 protocols using sc 55529

Measuring NF-κB and iNOS Protein Levels

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The expression levels of transcription factor NF-κΒ and iNOS protein in the liver homogenate were measured by immunoblotting. Briefly, 50 µg of protein was resolved on 10% SDS polyacrylamide gels, and then transferred to polyvinylidene fluoride membranes and blocked with 5% (w/v) nonfat milk in TBS-T (10 mM Tris/HCl, 150 mM NaCl (pH 7.6) and 0.05% TWEEN 20) for 1 h at 20 °C. Next, the membranes were incubated overnight at 4 °C on a shaker with primary anti- NF-κΒ (1:400; ab-7971, Abcam, Cambridge, UK), anti-NOS2 (1:200; sc-7271, Santa Cruz, CA, USA), or anti-β-tubulin (1:200; sc-55529, Santa Cruz, CA, USA). The samples were incubated for 2 h or overnight at 4 °C on a shaker with HRP-conjugated goat anti-rabbit IgG (1:4000; sc-2004, Santa Cruz, CA, USA) or m-IgGκ BP (1:1000; sc-516102, Santa Cruz, CA, USA). Images were acquired using chemiluminescence and a ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA), and were analyzed with ImageLab 6.0.1. (Bio-Rad, Hercules, CA, USA). The relative protein levels were calculated based on β-tubulin expression as a loading control.

Trejo-Hurtado C.M., Landa-Moreno C.I., la Cruz J.L., Peña-Montes D.J., Montoya-Pérez R., Salgado-Garciglia R., Manzo-Avalos S., Cortés-Rojo C., Monribot-Villanueva J.L., Guerrero-Analco J.A, & Saavedra-Molina A. (2023). An Ethyl Acetate Extract of Eryngium carlinae Inflorescences Attenuates Oxidative Stress and Inflammation in the Liver of Streptozotocin-Induced Diabetic Rats. Antioxidants, 12(6), 1235.

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Western Blot Analysis of β-Catenin Signaling

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SW480 cells at 1 × 10⁶ cells/mL were treated with different concentrations of inhibitors for 24 h. Cells were lysed in buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors. After centrifugation at 12,000 rpm for 20 min at 4 °C, the supernatant was loaded onto an 8% SDS polyacrylamide gel for electrophoretic analysis. Separated proteins were transferred onto nitrocellulose membranes for immunoblot analysis. The antibodies against total β-catenin (610153, BD Biosciences, most of which is phosphorylated β-catenin and represents the E-cadherin bound pool), the active form of β-catenin (ABC, 05–665, EMD Millipore, dephosphorylated at positions S37 and T41 of β-catenin), cyclin D1 (sc-853, Santa Cruz Biotechnology, Inc.), c-myc (D84C12, Cell Signaling), and β-tubulin (sc-55529, Santa Cruz Biotechnology, Inc.) were incubated with the membranes overnight at 4 °C, respectively. IRDye 680LT goat antimouse IgG (827–11080, LiCOR) or IRDye 800CW goat antirabbit IgG (827–08365, LiCOR) was used as the secondary antibody. The images were detected by the Odyssey Infrared Imaging System (LiCOR). Experiments were performed in duplicate.

Zhang M., Wang Z., Zhang Y., Guo W, & Ji H. (2018). Structure-Based Optimization of Small-Molecule Inhibitors for the β-Catenin/B-Cell Lymphoma 9 Protein–Protein Interaction. Journal of medicinal chemistry, 61(7), 2989-3007.

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Immunostaining of Reprogrammed Cells

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Cells were seeded in 48-wells plate coated with gelatin 0.1% (w/v). Immunofluorescence was performed at day 7 of the OSKM reprogramming process or at day 12 of transdifferentiation assay. Briefly, cells were fixed with 4% paraformaldehyde (w/v) for 15 min at room temperature for all antibodies except anti-β TUBULIN, where 100% methanol was used for 10 min at −20°C. After fixation, cells were blocked and permeabilized with 2% (w/v) BSA and 0.1% (v/v) Triton X-100 diluted in Dulbecco’s phosphate buffered saline (PBS) for 1 hour at RT. For immunostaining, cells were incubated overnight with primary antibodies against CDH1 (BD, 610182), SSEA1 (Thermo Fisher, MA1–022-D488), TUJ1 (GeneTex, GTX631836) and β-TUBULIN (Santa Cruz, sc-55529) in PBS with 2% BSA (w/v) and 0.1% Triton X-100 (v/v). The next day, cells were incubated with fluorophore-labeled secondary antibodies, together with 4’−6-diamidino-phenylindole (DAPI) diluted in PBS with 2% BSA (w/v) and 0.1% Triton X-100 (v/v) for 1 h at room temperature. Immunofluorescence images were taken with a Leica DMI 6000 inverted microscope at 4x or 40x magnification, and image settings were maintained at constant levels for each set of images acquired per factor stained.

Guallar D., Fuentes-Iglesias A., Souto Y., Ameneiro C., Freire-Agulleiro O., Pardavila J.A., Escudero A., Garcia-Outeiral V., Moreira T., Saenz C., Xiong H., Liu D., Xiao S., Hou Y., Wu K., Torrecilla D., Hartner J.C., Blanco M.G., Lee L.J., López M., Walkley C.R., Wang J, & Fidalgo M. (2020). ADAR1-dependent RNA editing promotes MET and iPSC reprogramming by alleviating ER stress. Cell stem cell, 27(2), 300-314.e11.

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Wnt3a-Induced Axin2 and Cyclin D1 Analysis

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SW480 cells at 1 × 10⁶ cells/mL were treated with different concentrations of inhibitors for 24 h. MDA-MB-231 cells at 1 × 10⁶ cells/mL were treated with Wnt3a (100 ng/mL) for 30 min, then the inhibitors at different concentration were added and incubated for 24 h. Cells were lysed in buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors. After centrifugation at 12 000 rpm for 20 min at 4 °C, the supernatant was loaded onto an 8% SDS polyacrylamide gel for electrophoretic analysis. Separated proteins were transferred onto nitrocellulose membranes for immunoblot analysis. The antibodies against Axin2 (MA5–15015, Thermo Fisher. Immunogen: residues surrounding Pro566 of human Axin2), cyclin D1 (sc-853, Santa Cruz Biotechnology. Immunogen: human cyclin D1 residues 1−295), and β-tubulin (sc-55529, Santa Cruz Biotechnology) were incubated with the membranes overnight at 4 °C. IRDye 680LT goat antimouse IgG (827–11080, LiCOR) or IRDye 800CW goat antirabbit IgG (827–08365, LiCOR) was used as the secondary antibody. The images were detected by the Odyssey Infrared Imaging System (LiCOR). Experiments were performed in triplicate.

Wang Z., Zhang M., Quereda V., Frydman S.M., Ming Q., Luca V.C., Duckett D.R, & Ji H. (2021). Discovery of an Orally Bioavailable Small-Molecule Inhibitor for the β-Catenin/B-Cell Lymphoma 9 Protein–Protein Interaction. Journal of medicinal chemistry, 64(16), 12109-12131.

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SH2B1 Protein Immunoblotting and Immunoprecipitation

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Frozen tissues were lysed in L-RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100, pH 7.2 containing 1 mM Na₃V0₄, 1 mM PMSF, 10 μg/mL aprotinin, 1 μg/mL leupeptin). Equal amounts of protein were immunoblotted with antibody to SH2B1 (αSH2B1) (sc-136065, RRID:AB_2301871; Santa Cruz Biotechnology) (1:1,000 dilution) or β-tubulin (sc-55529, RRID:AB_2210962; Santa Cruz Biotechnology) (1:1,000 dilution) as described in Joe et al. (19 (link)). For immunoprecipitations, tissue lysates containing equal amounts of protein were incubated with αSH2B1 (1:100) and immunoprecipitated and immunoblotted as in Joe et al. PC12 cells (ATCC) were cultured and treated as in Joe et al. Briefly, the cells were grown in PC12 medium A (RPMI medium, 5% FBS, 10% heparan sulfate) in 10-cm dishes coated with rat tail type I collagen (#354236; Corning). Cells were transfected and, 24 h later, incubated overnight in deprivation medium (RPMI medium, 2% heparan sulfate, 1% FBS) before being lysed and immunoblotted with αSH2B1.

Flores A., Argetsinger L.S., Stadler L.K., Malaga A.E., Vander P.B., DeSantis L.C., Joe R.M., Cline J.M., Keogh J.M., Henning E., Barroso I., Mendes de Oliveira E., Chandrashekar G., Clutter E.S., Hu Y., Stuckey J., Farooqi I.S., Myers MG J.r, & Carter-Su C. (2019). Crucial Role of the SH2B1 PH Domain for the Control of Energy Balance. Diabetes, 68(11), 2049-2062.

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Liver Protein Extraction and Western Blotting

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Pig liver was homogenized on ice in RIPA buffer (1% NP-40, 1% Na-deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M NaPO4 pH 8.0, 2 mM EDTA) supplemented with protease inhibitor cocktail (Sigma), 1 mM PMSF (Sigma), and 1 mM DTT. The homogenate was centrifuged at 4°C, 10,000×g for 30 minutes, and the supernatant was combined with SDS-PAGE sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 100 mM DTT, 0.1% bromophenol blue). The liver lysate samples were heated in boiling water for 5 minutes, cooled on ice, and sonicated with a Sonic Dismembrator (Fisher Scientific). The samples were electrophoresed in a 7.5% SDS-PAGE gel with Tris-Glycine running buffer. Protein was transferred onto a PVDF Immobilon-P Transfer Membrane (Millipore) using XCell II Blot Module (Invitrogen) according to manufacturer's instructions. Immunoblot analysis was performed using primary antibodies against LDLR (EP1553Y, Abcam), β–tubulin (SC-55529, Santa Cruz Biotechnology), and secondary antibodies conjugated with horseradish peroxidase (SC-2317 and SC-2005, Santa Cruz Biotechnology). Signal was detected using Chemiluminescent Detection Kit (Pierce).

Davis B.T., Wang X.J., Rohret J.A., Struzynski J.T., Merricks E.P., Bellinger D.A., Rohret F.A., Nichols T.C, & Rogers C.S. (2014). Targeted Disruption of LDLR Causes Hypercholesterolemia and Atherosclerosis in Yucatan Miniature Pigs. PLoS ONE, 9(4), e93457.

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Regulation of ETV4 by MED25 in PC3 Cells

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PC3 cells were obtained from American Type Culture Collection and cultured accordingly. Full-length MED25 cDNA was cloned into pQCXIH (Clontech) with an added C-terminal 3× FLAG tag. Oligonucleotide sequences for MED25 shRNA hairpin design are as follows, with targeting sequences in lower case: MED25a_fwd-CCGGtgattgagggtacggccaaTTCAAGAGAttggccgtaccctcaatcacaTTTTTG MED25a_rev-AATTCAAAAAtgtgattgagggtacggccaaTCTCTTGAAttggccgtaccctcaatca MED25b_fwd- CCGGtcaaaggcctctaccgcatTTCAAGAGAatgcggtagaggcctttgagaTTTTTG MED25b_rev- AATTCAAAAAtctcaaaggcctctaccgcatTCTCTTGAAatgcggtagaggcctttga MED25 shRNA hairpins were cloned into the pLKO.1 lentiviral expression vector [80 (link)], and expression and infection of lentivirus performed following standard protocol. ETV4 shRNA retroviral constructs were previously described, and retrovirus production and infections were carried out following previously published methodology [81 (link)]. Whole-cell extracts from cells expressing control shRNA constructs, ETV4 shRNA or MED25 shRNA were run on SDS-PAGE gels and blotted to nitrocellulose membranes following standard procedures. Antibodies used for immunodetection were ETV4, ARP32262 (Aviva Systems Biology); MED25, ARP50699_P050 (Aviva Systems Biology); and beta-Tubulin, sc-55529 (Santa Cruz).

Currie S.L., Doane J.J., Evans K.S., Bhachech N., Madison B.J., Lau D.K., McIntosh L.P., Skalicky J.J., Clark K.A, & Graves B.J. (2017). ETV4 and AP1 transcription factors form multivalent interactions with three sites on the MED25 activator-interacting domain. Journal of molecular biology, 429(20), 2975-2995.

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Extraction and Analysis of Pluripotent Stem Cell Proteins

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Whole cell extracts were obtained by lysing ESCs subjected to the different treatments with RIPA Lysis Buffer System (sc-24948; Santa Cruz Biotechnology) with freshly added protease and phosphatase inhibitors (sc-24948; Santa Cruz Biotechnology). Lysates were incubated during 30 min at 4°C and centrifuged for 10 min at 10,000g at 4°C to remove cellular debris. Samples were kept at −80°C until use.
Protein samples for Western blot analysis were prepared by adding Laemmli buffer and denaturing for 5 min at 95°C and loaded for resolution in a Novex WedgeWell 4–20% tris-Glycine Gel (XP04205BOX; Invitrogen). For Western blotting, the following antibodies and dilutions were used: αBMAL1 (1:2,000, ab93806; Abcam), αPOU5F1 (1:4,000, sc-5279; Santa Cruz Biotechnology), αNANOG (1:2,000, A300-397A-2; Bethyl Laboratories), αZFP42 (1:1,000, sc-514643; Santa Cruz Biotechnology), αEZH2 (1:5,000, #5246S; Cell Signaling Technology), αSUZ12 (1:1,000, sc-271325; Santa Cruz Biotechnology), and α-ACTIN (1:2,000, sc-47778; Santa Cruz Biotechnology) or β-TUBULIN (1:2,500, sc-55529; Santa Cruz Biotechnology) as loading controls.

Ameneiro C., Moreira T., Fuentes-Iglesias A., Coego A., Garcia-Outeiral V., Escudero A., Torrecilla D., Mulero-Navarro S., Carvajal-Gonzalez J.M., Guallar D, & Fidalgo M. (2020). BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells. Life Science Alliance, 3(5), e201900534.

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Western Blot Analysis of BACH1 Protein

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The tissues were lysed in RIPA buffer supplemented with protease and phosphatase inhibitor at 5 mg ml⁻¹ concentration. The supernatant containing proteins was collected after centrifuging tissue lysates at 12,000 r.p.m. at 4 °C. Protein concentration was determined by the BCA protein assay kit, and 20 µg of protein samples was boiled and loaded onto SDS–PAGE gels. The gels were transferred to 0.22-µm nitrocellulose membranes and blocked with 5% non-fat milk in 1× Tris-Buffered Saline containing 0.1% Tween 20 (TBST). The membranes were incubated with primary antibodies against BACH1 (Santa Cruz Biotechnology, sc-271211) and β-tubulin (Santa Cruz Biotechnology, sc-55529) at 4 °C overnight. After three washes (15 min, 5 min and 5 min) with 1× PBS containing 0.1% Tween 20 (PBST), the membranes were incubated with a rabbit secondary antibody conjugated with horseradish peroxidase (1:2,000) for 1 h, followed by three washes (15 min, 5 min and 5 min) with PBST. A chemiluminescence reagent kit was used to visualize protein bands with horseradish peroxidase secondary antibodies.

Wan Y., Cohen J., Szenk M., Farquhar K.S., Coraci D., Krzysztoń R., Azukas J., Van Nest N., Smashnov A., Chern Y.J., De Martino D., Nguyen L.C., Bien H., Bravo-Cordero J.J., Chan C.H., Rosner M.R, & Balázsi G. (2023). Nonmonotone invasion landscape by noise-aware control of metastasis activator levels. Nature Chemical Biology, 19(7), 887-899.

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Veliparib Modulates Retinal Injury in Mice

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For the veliparib treatment study, C57BL/6 mice with or without optic nerve crush injury were treated once daily i.p. with veliparib (10 mg/kg/d, cat. #A3002; ApexBio Technology) or the same volume of normal saline as vehicle for 5 d beginning on the day of injury. Retina were dissected and sonicated in radioimmunoprecipitation assay (RIPA) buffer and centrifuged at 20,000 × g for 30 min. The pellet and lysate were resolved by SDS-PAGE, transferred to nitrocellulose membranes, immunoblotted with anti-poly (ADP-ribose) (1:1000, cat. #4335-MC-100, RRID: AB_2572318, Trevigen), anti–β-actin (1:3000, cat. #8457, RRID: AB_10950489, Cell Signaling Technology), and anti–β-tubulin (1:2000, cat. #sc-55529, AB_2210962, Santa Cruz Biotechnology) primary antibodies. After primary antibody incubation, secondary antibodies (Odyssey IRDye 680 or 800) were applied for 1 h at room temperature. Membranes were then washed and visualized using a Licor Odyssey Infrared imaging system. For Parp1 mutant mice study, 129S-Parp1^tm1Zqw/J and 129S1/SvImJ mice retinas were analyzed by the same method as described above.

Wang X., Sekine Y., Byrne A.B., Cafferty W.B., Hammarlund M, & Strittmatter S.M. (2016). Inhibition of Poly-ADP-Ribosylation Fails to Increase Axonal Regeneration or Improve Functional Recovery after Adult Mammalian CNS Injury. eNeuro, 3(6), ENEURO.0270-16.2016.

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