R960 25

Manufactured by Merck Group

Sourced in United States

The R960-25 is a laboratory centrifuge capable of processing samples at high speeds. It is designed to separate the components of a mixture based on their density differences. The centrifuge can accommodate a variety of rotor types and sample volumes, making it a versatile piece of equipment for various laboratory applications.

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12 protocols using r960 25

Comprehensive Protein Extraction and Analysis

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Protein extraction from cells and tissues and analysis via SDS-PAGE and Western blotting were carried out as described previously (Sha et al., 2009 (link); Yang et al., 2010 (link)). Quantification of signals was done using BioRad ImageLab software. Glycosylation status was assessed by incubation with EndoH and PNGaseF enzymes for 1 h at 37°C (NEB). Total protein levels were normalized with the help of loading controls. The following primary antibodies were used: KI67 (1:200, Abcam ab16667); CYCLIN D1 (1:100 for staining, 1:2000 for Western blot, Abcam ab16663); c-MYC (1:1000, MilliporeSigma C3956); V5 (1:5000 for Western blot, 1:250 for immunoprecipitation, Invitrogen R960-25); HA (1:3,000, Sigma H9658); WNT5A (1:2000 for Western blot, 1:100 for immunoprecipitation, Proteintech 55184-1-AP); WNT5B (1:1000, Abclonal A8313); WNT1 (1:1000, Proteintech 27935-1-AP); E-CADHERIN (1:1000, BD 610181); α-TUBULIN (1:2,000, Santa Cruz sc-5286); HSP90 (1:1,000, Abcam ab13492); CALNEXIN (1:5000, Enzo ADI-SPA-860-F); SEL1L (1:2,000, Abcam ab78298); HRD1 (1:300, kindly gifted by Dr. Richard Wojcikiewicz, SUNY Upstate Medical University for Western blot; 1:1000, Sigma HPA024300 for immunostaining of human liver tumors). The following secondary antibodies were used: goat anti-mouse and goat anti-rabbit IgG-HRP (1:5,000; BioRad).

Bhattacharya A., Wei J., Song W., Gao B., Tian C., Wu S.A., Wang J., Chen L., Fang D, & Qi L. (2022). SEL1L-HRD1 ER-associated degradation suppresses hepatocyte hyperproliferation and liver cancer. iScience, 25(10), 105183.

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Protein Extraction and Western Blotting

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Cells were collected in ice-cold PBS and centrifuged at 150 x g at 4°C for 10 min. The cell pellets were re-suspended in 800 μl lysis buffer (HEPES 50mM, NaCl 80mM, Protease Inhibitor Cocktail 1%, TritonX 1% up to 10ml Millipore), incubated on ice for 30 min and sonicated 10 times for 10 sec, with 10 sec breaks on ice. After centrifugation at 10,500 x g for 10 min at 4°C, the supernatants were transferred to clean tubes. Purified lysates were mixed with NuPAGE-LDS Sample Buffer (4x, Invitrogen, NP0007) and Reducing agent (10x, Life Technologies, NP0004). The mixtures were boiled at 95°C for 5 min. After protein separation on 4–12% NuPAGE-Bis-Tris gels (Life Technologies, NP0322BOX) at 200V for 45 min, the proteins were blotted on PVDF membrane (Invitrogen) at 32V for 2 hrs. Western blotting was carried out using the WesternBreeze chemiluminescent kit (Invitrogen) according to manufacturer’s instructions. Immunoblotting was performed using mouse monoclonal anti-V5 (1:5.000, Thermo Fisher, R960-25) and anti-GAPDH (1:10.000, Sigma Aldrich, G8795) antibodies. Membranes were then exposed to an X-ray film for 5–10 min.

Schlaweck A.E., Tazi-Ahnini R., Ü. Basmanav F.B., Mohungoo J., Pasternack-Ziach S.M., Mattheisen M., Oprisoreanu A.M., Humbatova A., Wolf S., Messenger A, & Betz R.C. (2019). Autosomal-dominant hypotrichosis with woolly hair: Novel gene locus on chromosome 4q35.1-q35.2. PLoS ONE, 14(12), e0225943.

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Antibody Sources for Protein Detection

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Commercially available antibodies against DNAJA2 (rabbit monoclonal), DNAJB1 (mouse monoclonal), V5 tag (mouse monoclonal), GAPDH (mouse monoclonal), Pgk1 (mouse monoclonal) and mCherry (mouse monoclonal) were obtained from Abcam (UK)(ab157216; RRID:AB_2650527, Enzo life sciences (NY, USA) (ADI-SPA-450-E; RRID:AB_10621843), Invitrogen (CA, USA)(R960-25; RRID:AB_2556564), Sigma (G8795; RRID:AB_1078991), Invitrogen (459250; RRID:AB_2532235) and Abcam (ab125096; RRID:AB_11133266), respectively. Anti-mouse Ydj1 (SMC-150; RRID:AB_2570364) and anti-rabbit Sis1 (COP-080051; RRID:AB_10709957) were obtained from StressMarq Biosciences Inc. (Canada), and Cosmo Bio Co. (Japan), respectively. Antibody against YFP (rabbit polyclonal; RRID: AB_2650530) was generated in the laboratory. Anti-DnaK (rabbit polyclonal; RRID:AB_2650528) and anti-human Hsp/Hsc70 (rabbit polyclonal; RRID:AB_2650529) antibodies were a kind gift from Matthias Mayer (University of Heidelberg).

Nillegoda N.B., Stank A., Malinverni D., Alberts N., Szlachcic A., Barducci A., De Los Rios P., Wade R.C, & Bukau B. (2017). Evolution of an intricate J-protein network driving protein disaggregation in eukaryotes. eLife, 6, e24560.

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Protein Extraction and Western Blot Analysis

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Muscle tissue of 6‐ to 25‐week‐old mice was lysed in RIPA buffer (50 mM Tris–HCl pH7.4, 150 mM NaCl, 1% NP‐40, 0.25% Na‐Deoxycholate, 1× cOmplete Protease Inhibitor cocktail, Roche #4693132001) by sonication. HEK 293 cells were lysed as described for the RNA–protein pull‐down. 10–40 mg of protein extracts was separated using a 9% Bis‐Tris SDS–PAGE for muscle tissue protein extracts and precast protein gels (Thermo #NBP0321BOX) for cellular protein extracts. Proteins were blotted onto nitrocellulose membranes. The following antibodies were used after blocking membranes with 3% BSA in TBS‐T: Anti‐RuvBl1 (1:1,000; Proteintech #10210‐2‐AP), anti‐RuvBl2 (1:1,000; Bethyl #A302‐536A), anti‐TFPT (1:500; Proteintech#10097‐2‐AP), anti‐WDR5 (1:1,000; Bethyl #A302‐430A), anti‐YY1 (1:1,000; Cell signaling #46395), RalA (1:5,000; Becton Dickinson #R23520), anti‐V5 (1:5,000; Invitrogen #R960‐25), and anti‐FLAG (1:1,000, Sigma‐Aldrich #F1804). Secondary antibodies were anti‐mouse‐HRP (1:5,000, Thermo #31450) and anti‐rabbit‐HRP (1:5,000, Thermo #31460).

Schutt C., Hallmann A., Hachim S., Klockner I., Valussi M., Atzberger A., Graumann J., Braun T, & Boettger T. (2020). Linc‐MYH configures INO80 to regulate muscle stem cell numbers and skeletal muscle hypertrophy. The EMBO Journal, 39(22), e105098.

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Western Blot Antibody Validation

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Antibodies are listed as follows: p-Akt (1:1,000, Cell Signaling, 4056), Akt (1:1,000, Cell Signaling, 9272), p-eIF2α (1:1,000, Cell Signaling, 9721), p-IRE1α -S724 (1:1,000, Novus Biologicals, NB100-2323), GFP (1:10,000, Abclonal, AE012), poly-ubiquitination (1:1,000, FK1 clone, Enzo, BML-PW8805), V5 (1:5,000, Invitrogen, R960-25), and α-Tubulin (1:8,000, Sigma, T6199). The secondary antibodies used are as follows: goat anti-rabbit IgG-HRP (1:3,000, Santa Cruz) and goat anti-mouse IgG-HRP (1:3,000, Santa Cruz).

Zhao P., Huang P., Xu T., Xiang X., Sun Y., Liu J., Yan C., Wang L., Gao J., Cui S., Wang X., Zhan L., Song H., Liu J., Song W, & Liu Y. (2021). Fat body Ire1 regulates lipid homeostasis through the Xbp1s-FoxO axis in Drosophila. iScience, 24(8), 102819.

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Protein Extraction and Immunoblotting Protocol

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Cell extracts were prepared in UTB buffer (50 mM Tris–HCl pH 7.5, 150 mM β-mercaptoethanol, 8 M urea) and sonicated to release DNA-bound proteins. Primary antibodies used were rabbit anti-RAD51 (Abcam, ab63801, 1:1,000), rabbit anti-EXO1 (Bethyl, A302-640, 1:1,000), rabbit anti-PRIMPOL (described in ref. ^{11 (link)}, 1:1000), rabbit anti-SMC1 (a kind gift from A. Losada’s lab, CNIO^{59 (link)}, 1 μg/ml), mouse anti-V5 tag (Invitrogen, R960-25, 1:1:500), and mouse anti-αTUBULIN (B512, Sigma T6074, 1:10,000).

Piberger A.L., Bowry A., Kelly R.D., Walker A.K., González-Acosta D., Bailey L.J., Doherty A.J., Méndez J., Morris J.R., Bryant H.E, & Petermann E. (2020). PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts. Nature Communications, 11, 5863.

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Western Blot Analysis Protocol

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For all the gels shown in this study, proteins separated on NuPAGE 4-12% Bis- Tris gels were transferred to nitrocellulose membrane. Media and plasma samples were further incubated with Ponceau S solution to ensure equal loading and washed with PBST (PBS, 0.05% Tween 20). Blots were blocked in Odyssey blocking buffer for 30 min at room temperature and incubated with primary antibodies (Mouse anti-V5 antibody, Invitrogen R960-25, 1:1000 dilution; Mouse anti-Flag antibody (Sigma F1804), 1:1000 dilution; Rabbit anti-beta Tubulin antibody (Abcam ab6046), 1:5000 dilution; Rabbit anti-beta-Actin (Abcam ab8227), 1:5000 dilution; Rabbit anti-BHMT antibody (Abcam 96415); in blocking buffer for 1 h at room temperature or overnight at 4°C. After washing three times with PBST, blots were stained with species matched secondary antibodies (Streptavidin-AlexaFluor680, ThermoFisher S32358, 1:1000 dilution; Goat anti-mouse IRDye 680RD, LI-COR 925-68070, 1:10000 dilution; Goat anti-rabbit IRDye 800RD, LI-COR 925-68070, 1:10000 dilution. Blots were then washed three times with PBST and imaged with Odyssey CLx Imaging System.

Wei W., Riley N.M., Yang A.C., Kim J.T., Terrell S.M., Li V.L., Garcia-Contreras M., Bertozzi C.R, & Long J.Z. (2020). Cell type-selective secretome profiling in vivo. Nature chemical biology, 17(3), 326-334.

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Immunofluorescence Staining Protocol

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For staining, cells were washed in saline containing phosphate-buffered saline (PBS; BI), fixed in formaldehyde (4%), and permeabilized in PBS with 0.1% Triton X-100. Next, blocking in blocking solution (Sigma) was performed for 30 min, followed by incubation with a primary antibody in antibody diluent containing FBS (Sigma), and then with Alexa fluorophore-conjugated secondary antibodies (Alexa Fluor 488, Alexa Fluor 594) and DAPI. Z- stack images were taken using an LSM 800 (Zeiss) confocal microscope with 40/60× objective oil immersion.
The following antibodies were used for IF: p62 (Santa Cruz D-3), p53 (DO-1, Santa Cruz SC-126 and CM1, BioLegend-SIG-3520), Connexin 43 (Abcam, ab11370), V5 (ThermoFisher, R960-25), and FLAG (Merck-Sigma, F1804). F-actin was visualized with phalloidin-fluorescein isothiocyanate (FITC) (Sigma P5282), ZYX (kindly provided by Prof. Benjamin Geiger, made by Antibody Production Laboratory of the Department of Biological Services, Weizmann Institute of Science), anti-K48 (Merck, 05-1307), and anti-K63 (Merck, 05-1308). Monoclonal anti-p115 was kindly provided by Prof. Sima Lev (Weizmann Institute of Science, Israel).

Mukherjee S., Maddalena M., Lü Y., Martinez S., Nataraj N.B., Noronha A., Sinha S., Teng K., Cohen-Kaplan V., Ziv T., Arandkar S., Hassin O., Chatterjee R., Pirona A.C., Shreberk-Shaked M., Gershoni A., Aylon Y., Elazar Z., Yarden Y., Schramek D, & Oren M. (2022). Cross-talk between mutant p53 and p62/SQSTM1 augments cancer cell migration by promoting the degradation of cell adhesion proteins. Proceedings of the National Academy of Sciences of the United States of America, 119(17), e2119644119.

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Western Blot Analysis Protocol

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Western Blot Analysis of Protein Interactions

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Protein lysates harvested from mouse tissue, HEK293 cell lysate and immunoprecipitations were subjected to reducing SDS-PAGE analysis. Proteins in polyacrylamide gels were transferred to nitrocellulose membranes, which were then probed with the following antibodies: anti-myc (Santa-Cruz, 9E10, sc-40, HRP-conjugated, 1:500); anti-V5 (Invitrogen, R960-25, 1:2000); anti-FLAG (Sigma, F1804, 1:500); anti-LOX (LSBio, LS-C143168, 1:3000); and anti-ADAMTSL2 (GeneTex, GTX102069, 1:500). Equal protein loading was determined using an antibody directed against P97 (1:3000; kindly provided by Ariel Stanhill, Technion, IL) or actin (MP Biochemicals; clone C4, 1:5000).

Aviram R., Zaffryar-Eilot S., Hubmacher D., Grunwald H., Mäki J.M., Myllyharju J., Apte S.S, & Hasson P. (2018). Interactions between lysyl oxidases and ADAMTS proteins suggest a novel crosstalk between two extracellular matrix families. Matrix biology : journal of the International Society for Matrix Biology, 75-76, 114-125.

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