For Cdc20 and BubR1 immunoprecipitation, 2 μg of plasmid was transfected into HeLa cells in a 15 cm dish 48 h before collection. Cells were synchronized by double thymidine arrest followed by overnight treatment by nocodazole (200 ng/ml). Mitotic cells were shaken off the plate and lysed on ice in a lysis buffer containing 10 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, and 0.5% NP40 with protease and phosphatase inhibitors (Roche). The cell lysate was centrifuged at 17,000 g for 10 min at 4 °C, and the supernatant was applied to 20 μl of GFP-Trap beads (Chromotek) and shaken for 2 h at 4 °C. The beads were washed three times with 0.5 ml lysis buffer and boiled in 50 μl 2× SDS loading buffer. For Cdc20 knocking out examination, cells from a 10 cm dish were collected and lysed in 200 μl of lysis buffer as described above. The cell lysate was cleaned by centrifugation and boiled in an SDS loading buffer. A quantitative western blot (Odyssey DLx, LI-COR) was performed to examine Cdc20 and interested proteins. Antibodies used include Cdc20 (Santa Cruz, sc-13162; Bethyl, A301-180A), BubR1 (homemade in JN lab), Mad1 (Sigma, M8069), Mad2 (homemade in JN lab), Apc7 (Santa Cruz, sc-365649), Apc15 (Santa Cruz, sc-398488) and GAPDH (Proteintech, 60004-1). Fluorophore-labeled secondary antibodies include goat anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680CW (LI-COR).
Odyssey dlx
Manufactured by LI COR
Sourced in United States
The Odyssey DLx is a near-infrared western blot detection system designed for quantitative analysis of protein expression. It utilizes fluorescent dye-labeled secondary antibodies and near-infrared imaging technology to provide sensitive and accurate protein quantification.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Intercept blocking buffer, by LI COR (2 mentions)
Agt 001, by Synaptic Systems (2 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Anti β actin c4, by Santa Cruz Biotechnology (2 mentions)
Anti granzyme k, by Thermo Fisher Scientific (2 mentions)
Anti phospho stat3 tyr705 d3a7, by Cell Signaling Technology (2 mentions)
Anti phospho p44 42 mapk erk1 2 thr202 tyr204, by Cell Signaling Technology (2 mentions)
Image studio, by LI COR (2 mentions)
Irdye 680rd donkey anti mouse igg, by LI COR (2 mentions)
Irdye 800cw donkey anti rabbit igg, by LI COR (2 mentions)
Gfp trap beads, by Proteintech (1 mentions)
Irdye 800cw goat anti mouse, by LI COR (1 mentions)
Goat anti rabbit irdye 680cw, by LI COR (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Gapdh, by Proteintech (1 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Micro bca protein assay, by Thermo Fisher Scientific (1 mentions)
21 protocols using odyssey dlx
1
Immunoprecipitation and Quantitative Western Blot Analysis of Mitotic Regulators
2
Studying Ubiquitination Regulation by CCT5
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In vitro ubiquitination reactions were set by mixing 70 nM wild‐type or mutant biotinylated DDB1‐DCAF12 with 70 nM CUL4B‐RBX1 purified as previously described (Slabicki et al, 2020 (link)) in the presence or absence of 500 nM CCT5 or 250 nM TRiC (which contains two copies of CCT5) in a reaction mixture containing a 50 nM E1 enzyme (UBA1, Boston Biochem), a 1 μM E2 enzyme (UBCH5α, Boston Biochem) and 20 mM ubiquitin. Reactions were carried out in 50 mM Tris pH 7.5, 200 mM NaCl, 5 mM MgCl2, 0.2 mM CaCl2, 0.5 mM TCEP, 1 mM ATP, 0.1% Triton X‐100, 0.1 mg/ml BSA and 10% v/v glycerol and incubated for 0–30 min at 30°C. Reactions were then analyzed by Western blot on 0.2 μm nitrocellulose membranes using a mouse anti‐CCT5 primary antibody (Santa Cruz Biotechnology, sc‐376188, 1:5,000) and an Alexa Fluor 790‐labeled anti‐mouse secondary antibody (Invitrogen, #A11375, 1:10,000) using an Odyssey DLx (LiCor Biosciences).
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In vitro ubiquitination reactions were set by mixing 70 nM wild‐type or mutant biotinylated DDB1‐DCAF12 with 70 nM CUL4B‐RBX1 purified as previously described (Slabicki et al, 2020 (link)) in the presence or absence of 500 nM CCT5 or 250 nM TRiC (which contains two copies of CCT5) in a reaction mixture containing a 50 nM E1 enzyme (UBA1, Boston Biochem), a 1 μM E2 enzyme (UBCH5α, Boston Biochem) and 20 mM ubiquitin. Reactions were carried out in 50 mM Tris pH 7.5, 200 mM NaCl, 5 mM MgCl2, 0.2 mM CaCl2, 0.5 mM TCEP, 1 mM ATP, 0.1% Triton X‐100, 0.1 mg/ml BSA and 10% v/v glycerol and incubated for 0–30 min at 30°C. Reactions were then analyzed by Western blot on 0.2 μm nitrocellulose membranes using a mouse anti‐CCT5 primary antibody (Santa Cruz Biotechnology, sc‐376188, 1:5,000) and an Alexa Fluor 790‐labeled anti‐mouse secondary antibody (Invitrogen, #A11375, 1:10,000) using an Odyssey DLx (LiCor Biosciences).
3
In-cell Western Analysis of ER Expression
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In-cell Western analysis was performed in a similar manner as previously described34 (link). Briefly, T47D or MCF7:WS8 breast cancer cells were grown in blackout 96-well plates starting at 3000 cells per well. After 24 h, cells were placed in serum-starved media and allowed to acclimate for 48–72 h. Cells were treated with hormone, SERM, or SERD alongside vehicle control for 24 h. Only interior wells were used to avoid edge effects. Cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100. Plates were blocked with Intercept blocking buffer (LI-COR). Santa Cruz F10 anti-ERα antibody (sc-8002) was used as the primary at 1:200-500 dilution. Secondary antibody was a goat anti-mouse IgG 800 IRDye antibody (926-32210) from LI-COR. Cells were also treated with CellTag700 (926-41090) to control for differences in cell count between wells. All data were collected on a LI-COR Odyssey DLx and analyzed using Emperia Studio. Each experiment was comprised of three replicates.
4
Quantitative Protein Analysis of Immunoisolates
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Protein concentrations of all fractions from the immunoisolation were determined using the microBCA protein assay (Thermo Fisher Scientific #23235) following the manufacturer’s recommendations. For SDS-PAGE, 1 volume membrane sample buffer (8 M urea, 0.1 M Tris-HCl, pH 6.8, 5 mM EDTA, 3.2% (w/v) SDS, 0.05% (w/v) bromphenol blue, 4% [v/v] glycerol, and 4% (v/v) β-mercaptoethanol) was mixed with two volumes of the immunoisolation fraction, incubated at 60 °C for 10 min, and loaded onto 4–15% mini-PROTEAN TGX precast protein gels (Bio-Rad). Proteins were separated at 185 V for 35 min. After separation, proteins were transferred by semi-dry Western blotting onto nitrocellulose membranes (Amersham Protran Premium 0.45 µm). The proteins of interest were detected using specific primary antibodies and fluorescent secondary antibodies (see “Reagents and tools table”) on a fluorescence imager (LI-COR, Odyssey DLx). Signal intensities on immunoblots were quantified using ImageStudioLite.
5
Western Blot Procedure for 3T3 Cells
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Cells were lysed in 3T3 buffer (50 mM NaCl, 50 mM NaF, 30 mM sodium pyrophosphate, 25 mM HEPES, 2.5 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100; pH 7.5). Equal protein concentrations were separated via SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked in 5% (w/v) milk powder in TBS-T for 1 h prior to primary and secondary antibody (Supplementary Materials ) incubations and imaged via Odyssey DLx (LICOR).
6
Quantifying Phage Synthesis via Dot Blot
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2 µL of the diluted TXTL samples were blotted on a nitrocellulose membrane and let dry for 15 minutes. The membrane was incubated for 1 h with blocking buffer (TBST 1×, 5% w/v non-fat dry milk) on a shaker at RT. The membrane was rinsed 2 times with water and incubated for 1 hour on a shaker at room temperature (RT) with the primary antibody (T7 tag, PA1-32386, Thermofisher) diluted in the blocking buffer (1:10,000). The membrane was washed 3 times with the wash buffer (TBST 1×) and incubated for 1 h on a shaker at RT with the secondary antibody (IRDye® 800CW Goat anti-Rabbit IgG, LI-COR) diluted (1:20,000) in the blocking buffer. Finally, the membrane was washed 3 times with the wash buffer and scanned for fluorescence with an LI-COR Odyssey DLx. The calibration curve showed that the concentration of synthesized phages from the experiment was in the linear range of detection (Supplementary Fig. 10 ). For each experiment day (n = 3), the time points for the s-µg and the 1-g conditions were blotted on the same membrane along with an internal standard (bacteriophage T7 synthesized in TXTL at 1 nM DNA concentration). Dot blots of the three experiment days were performed on three consecutive days and analyzed in parallel by normalization to the internal standard.
7
HEK-293T Cell-Based Co-Immunoprecipitation
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We heterologously expressed pCMV6 constructs of each TMEM163 and ZNT protein using HEK-293T cells. The cells were lysed the next day and processed for co-IP using the Pierce Co-IP kit according to the manufacturer's protocol (see Appendix A SupportingInformation for details). All immunoblots were imaged using the Odyssey SA™ IR or Odyssey DLX™ imaging system (LI-COR Biosciences). WB images were processed using Adobe Photoshop 2022 to crop unwanted edges or empty lanes, equalize contrast levels, and straighten vertical position. We used Adobe Illustrator 2022 to compile and label images.
8
Westerns for Transfected HEK293T Cells
9
Isolation and Characterization of Extracellular Vesicles
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Extracellular vesicles (EV) were isolated from a subset of HCM patients (n = 12) and healthy volunteers (n = 8) plasma samples and from iPSC-derived cardiomyocyte supernatants [37 (link)]. The purity of EV from both plasma and supernatant samples was tested by Western blotting in reducing conditions [36 (link)]. Total protein extracts (20 µg) were loaded on an 8–20% acrylamide gel, then transferred to a PVDF membrane and incubated in Intercept blocking buffer for 1 h at room temperature (LI-COR Biosciences, Lincoln, NE, USA) followed by primary antibodies against ALG-2 interacting protein X (Alix, part of Endosomal Sorting Complexes Required for Transport [38 (link)], ab186429, Abcam, Cambridge, UK), tumor susceptibility gene 101 (TSG101, an ESCRT component [39 (link)], ab30871 Abcam, Cambridge, UK) and tetraspanin CD81 [40 (link)] (MA5-17937, Thermo Fisher Inc., Waltham, MA, USA). After washing, the membranes were incubated for 1 h with secondary antibody (anti-mouse IgG IRDye-conjugated, LiCor). Images were acquired by Odyssey DLx and analyzed by Image Studio software (LI-COR Biosciences, Lincoln, NE, USA).
10
Western Blot Analysis of Protein Acetylation
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Protein samples were resolved on 7–15% gradient polyacrylamide (SDS-PAGE) gels and transferred to nitrocellulose membranes via Trans Blot Turbo (Bio-Rad). Membranes were then blocked with 5% milk in Tris-buffered saline and 0.05% Tween 20 (TBST) for 45min at RT, incubated with primary antibodies diluted in 5% BSA in TBST and 0.1% NaN3 overnight at 4 °C with rocking, washed with TBST 3 times, and then secondary stained with 5% milk in TBST (for HRP-conjugated) or Intercept (TBS) blocking buffer (Li-Cor) supplemented with 0.2% Tween-20 (for IRDye-conjugated). Following 3 TBST washes and a TBS wash, blots were imaged on a Li-Cor Odyssey DLx (for IRDye-conjugated) or detected with chemiluminescence substrate (Li-Cor) and imaged on an Azure C600 CDD Imager (for HRP-conjugated). Protein signals were analyzed in FIJI by a fixed rectangular ROI around the band of interest. The local background for each lane was measured by ROI and subtracted. For acetylation of SMC3 (Fig. 5B ), both SMC3 and acetylated SMC3 signals were normalized to b-tubulin, and then the amount of normalized acetylated SMC3 was proportioned to SMC3.
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