Lipopolysaccharides (0111:B4) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies specific for GST (sc-459) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies specific for GRP78 (ab32618), CD14 (ab182032), and TLR4 (ab22048) were obtained from Abcam (Cambridge, UK). Antibodies specific for interferon regulatory transcription factor (IRF)3 (4302) and IRF3 phosphorylated at Ser396(4947) were obtained from Cell Signaling Technology (Danvers, MA, USA). F(ab′)2-goat anti-mouse Alexa Fluor®555 (AF555) was purchased from Thermo Fisher Scientific. Anti-TLR4-PE was obtained from PharMingen (Becton Dickinson, Franklin Lakes, NJ, USA). Anti-LAMP1-PE, anti-CD14-PECy7, and permeabilization buffer were obtained from eBioscience (San Diego, CA, USA).
Sc 459
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-459 is a laboratory instrument designed for the separation and purification of biological samples. It utilizes centrifugation technology to facilitate the isolation of specific cellular components or macromolecules from complex mixtures. The core function of Sc-459 is to provide researchers with a reliable and efficient tool for various analytical and preparative applications.
Automatically generated - may contain errors
Lab products found in correlation
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Ab182032, by Abcam (1 mentions)
Ab22048, by Abcam (1 mentions)
Ab32618, by Abcam (1 mentions)
β actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
Anti tlr4 pe, by BD (1 mentions)
Anti cd14 pecy7, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (1 mentions)
F1804, by Merck Group (1 mentions)
Irdye 800, by LI COR (1 mentions)
T6074, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
H1029, by Merck Group (1 mentions)
Tnt quick coupled transcription translation system, by Promega (1 mentions)
Sc 816, by Santa Cruz Biotechnology (1 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (1 mentions)
Rabbit anti gst, by Santa Cruz Biotechnology (1 mentions)
R960 25, by Thermo Fisher Scientific (1 mentions)
Nb100 122, by Novus Biologicals (1 mentions)
Sc 10790, by Santa Cruz Biotechnology (1 mentions)
12 protocols using sc 459
1
Lipopolysaccharide-Induced Signaling Pathway Analysis
2
Western Blot Antibody Optimization
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Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were sequentially incubated with primary and secondary antibodies The primary antibodies were used at the following dilutions: MYC (1:1,000; 2276 (9B11); Cell Signaling Technology), His (1:12,500; H1029; Sigma-Aldrich), FLAG (1:2,000; F1804; Sigma-Aldrich), GST (1:1,000; sc-459; Santa Cruz) α-tubulin (1:2,500; T6074; Sigma-Aldrich), Gβ (1:1,000; sc-261; Santa Cruz), Vangl2 (1:500; from S. Sokol; Ossipova et al., 2015 (link)), and p114RhoGEF (1:1,000; PA5-21429; Thermo Fisher Scientific). The secondary antibodies were goat anti-rabbit Alexa Fluor 680 (1:10,000; A21077; Invitrogen) and goat anti-mouse IRDye 800 (1:10,000; 926-32210; Li-Cor). Infrared imaging of immunoblots was performed using an Odyssey Infrared Imaging System (Li-Cor Biosciences). Images were processed using the ImageJ software and assembled for presentation using Photoshop and Illustrator.
3
GST-Fusion Protein Interaction Assay
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The GST and GST fusion proteins were expressed in Escherichia coli BL21(DE3) cells. Further purification was performed using glutathione-Sepharose 4B beads (GE Healthcare) according to the manufacturer's procedures. For in vitro AR protein synthesis, pSG5-AR plasmid was incubated with TNT quick-coupled transcription/translation system (Promega, Madison, WI, USA) master mix. Equal amounts of in vitro synthesised AR protein were incubated with purified GST-BCAS2 and GST-ARA70 (321–441) proteins in IP buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100). GST was used as a negative control. Interaction proteins were eluted and resolved by SDS–PAGE. Western blotting was performed with rabbit anti-AR (Santa Cruz, Dallas, TX, USA; sc-816) and rabbit anti-GST (Santa Cruz; sc-459) antibodies, respectively.
4
Immunoprecipitation and Immunoblotting Assay for HIF-1α, HIF-2α, and p300 Proteins
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Whole cell lysates were prepared in modified RIPA buffer [50 mM Tris-HCl (pH 7.5), 1 mM β-mercaptoethanol, 150 mM NaCl, 1% Igepal, and protease inhibitor cocktail] and incubated overnight with the following antibodies (catalog number and supplier): HIF-1α (sc-10790, Santa Cruz); V5 epitope (NB600-381, Novus Biologicals), HIF-2α (NB100-122, Novus Biologicals), or p300 (NB500-161, Novus Biologicals) in the presence of protein A Sepharose beads (NBP1-97240, Novus Biologicals). After washing three times, the bound proteins were fractionated by SDS-PAGE, followed by immunoblot assays using antibodies against the following: HIF-1α (610958, BD Bioscience); HIF-2α (NB100-122, Novus Biologicals); and V5 epitope (R960-25, Invitrogen). Other antibodies used in immunoblot assays recognized PRDX2 (H00007001-M01, Novus Biologicals) and PRDX4 (NBP2-19778, Novus Biologicals), and GST (sc-459, Santa Cruz) and actin (sc-1616, Santa Cruz).
5
Quantitative Immunoblotting of Cellular Proteins
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Quantitative immunoblotting was performed on proteins separated on 12% SDS-PAGE gels by transferring them overnight onto Nitrocellulose membrane. Membranes were incubated in primary antibodies against Astrin (Proteintech; 14726–1-AP; 1:3000), γ-Tubulin (Sigma-Aldrich; T6793; 1:800), PP1 (Santa-Cruz; sc-7482; 1:5000) and GST (Santa-Cruz; sc-459; 1: 500) for 2 hr and probed using secondary antibodies labelled with infrared fluorescent dyes, which were imaged using an Odyssey imager.
6
Annexin A2 Protein Identification Protocol
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A Donkey anti-goat IgG-HRP (sc-2033), a goat polyclonal antibody against AnnexinA2 (sc-1924), and a rabbit polyclonal antibody against GST (sc-459) were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Rabbit polyclonal anti-AnnexinA2 (ab75932) and goat anti-rabbit HRP (ab6721) were from Abcam (Cambridge, MA, USA). Rabbit polyclonal antibody against His6, Alexa fluor 488 labelled donkey anti-goat IgG(H + L) and alexa fluor 594 labelled donkey anti-rabbit IgG(H + L) were purchased from Invitrogen (Carlsbad, CA, USA). Plasmids were extracted using a Qiagen mini-prep kit from Qiagen (Mississauga, ON, Canada). PCR products were purified with a Qiagen PCR purification kit (Qiagen). All restriction enzymes were purchased from New England Biolab (Mississauga, ON, Canada). Fibrinogen, T4 DNA ligase, calf intestinal alkaline phosphatise and Taq polymerase were purchased from Invitrogen (Burlington, ON, Canada). LC/MS for protein identification was carried out at the McGill University and Genome Quebec Innovation Centre.
7
Western Blot Analysis of TGIF2LX Protein
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Western blot analysis was conducted as previously described (Raoofian et al, 2013). Briefly, cell lysates were subjected to 12% SDS–PAGE electrophoresis and transferred to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). The membranes were blocked for 1 h with 3% bovine serum albumin (BSA) at 37°C and then incubated with a 1:500 dilution of a polyclonal rabbit anti-TGIF2LX antiserum raised against the C-terminus (sc-459, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (2 h, at room temperature). The membranes were then incubated with 1:5,000 dilution of a goat HRP-conjugated anti rabbit IgG secondary antibody (1.5 h) followed by development with 4-chloro-1-naphthol (Immun-Blot, Bio-Rad Laboratories, Hercules, CA, USA).
8
Western Blot Analysis of TLS and GST
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Western blot analysis was performed as previously described [23 (link)]. Briefly, the membranes were incubated with anti-TLS (BD bioscience, 611385) or anti-GST antibody (Santa Cruz sc-459) for 1 h at room temperature. Then the membranes were washed with PBST for 5 min, four times, and incubated with anti-mouse HRP-conjugated IgG (Dako, P0161) or anti-rabbit HRP-conjugated HRP (Cell signaling, 70745). All the antibodies were diluted by 1 % skim milk to 1:2000. The signals were detected with SuperSignal West Pico substrate (Thermo Scientific).
9
Immunofluorescence and Immunoblot Antibodies
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The following antibodies were used for immunofluorescence and immunoblot: Anti-Mad2 (sc-65492, Santa Cruz Biotechnology, IF 1:75), Bub1 (K0168-3, MBL, IF 1:100, IB 1:1,000), CAP-H (sc-101013, Santa Cruz Biotechnologies, IB 1:1,000), Histone H2A phospho Thr 12032 (link) (IF 1:50), FLAG (012-22384, Wako, IF 1:1,000 or F7425, Sigma, 1:500), SET/TAF1β50 (link) (KM1720, IF 1:50, IB 1:2,000), hSgo251 (link) (IF 1:2,000 for Fig. 2 A and 1:5,000 for Fig. 2 F and L, IB 1:1,000), Aurora B (611,082, BD, IF 1:100, IB 1:1,000), pAurora A (T288)/B (T232) (2914S, Cell Signaling, IF 1:50, IB 1:2,000), Hec1 phospho Ser 55 (GTX70017, Gene Tex, IF 1:10,000), cyclin B1 (sc-245, Santa Cruz Biotechnology, IB 1:500), α-tubulin (T9026, Sigma, IF 1:5,000, IB 1:10,000), β-tubulin (T4026, Sigma, IF 1:1,000), GST (sc-459, Santa Cruz Biotechnology, IB 1:1,000) and Cenp-C (PD030, MBL, IF 1:1,000) antibodies. A For immunofluorescence, secondary antibodies (Invitrogen Alexa Fluor) were used at 1:1,000. For immunoblotting, secondary antibodies were used at 1:2,000.
10
Purification of GFP-Yck2p by GST-Sec2p
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Yeast cells overexpressing GFP-Yck2p (NY3138) were resuspended in lysis buffer containing 1× PBS, 1 mM MgCl2, 1 mM PMSF, protease inhibitors (Complete EDTA-free protease inhibitor cocktail tablets), and 5 mM DTT. Cells were disrupted in a bead beater using 0.5-mm zirconia/silica beads. Triton X-100 was added to the lysate to a final concentration of 1%, followed by a 15-min incubation at 4°C and finally clearance by centrifugation at 13,000 rpm for 20 min.
One hundred OD595 units of yeast lysate were incubated with 0.2 μM GST-Sec2p protein purified from bacteria and immobilized on glutathione–Sepharose beads. After 2 h of incubation at 4°C, the beads were washed quickly three times with 1 ml of a buffer containing 1× PBS, 5 mM MgCl2, 0.05% Triton X-100, and 1 mM DTT. Bound products were separated by SDS–PAGE and analyzed with anti-GFP monoclonal antibody (1:1000 dilution; sc-9996; Santa Cruz Biotechnology, Santa Cruz, CA) as the primary antibody. The amount of GST-Sec2p in the reaction was verified by Western blotting with anti-GST antibody (1:1000 dilution; sc-459; Santa Cruz Biotechnology). HRP-conjugated goat anti-mouse and anti-rabbit IgG (1:10,000 dilution) were used, respectively, as the secondary antibodies and detected with Pierce ECL Western Blotting Substrate.
One hundred OD595 units of yeast lysate were incubated with 0.2 μM GST-Sec2p protein purified from bacteria and immobilized on glutathione–Sepharose beads. After 2 h of incubation at 4°C, the beads were washed quickly three times with 1 ml of a buffer containing 1× PBS, 5 mM MgCl2, 0.05% Triton X-100, and 1 mM DTT. Bound products were separated by SDS–PAGE and analyzed with anti-GFP monoclonal antibody (1:1000 dilution; sc-9996; Santa Cruz Biotechnology, Santa Cruz, CA) as the primary antibody. The amount of GST-Sec2p in the reaction was verified by Western blotting with anti-GST antibody (1:1000 dilution; sc-459; Santa Cruz Biotechnology). HRP-conjugated goat anti-mouse and anti-rabbit IgG (1:10,000 dilution) were used, respectively, as the secondary antibodies and detected with Pierce ECL Western Blotting Substrate.
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