For the substrate ubiquitination assay, the purified Flag-p53, with the wild-type or the mutant E6, was incubated with 60 nM E1 (UBE1), 400 nM E2 (UbcH7), 400 nM E6AP, and 30 μM ubiquitin in ubiquitination reaction buffer. The reactions at 37 °C were initiated by the addition of ATP and quenched by mixing the reaction mixture with protein loading dye (Sangon Biotech) at the indicated time points. Samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblotting (IB) according to standard laboratory procedures, as described below. The unmodified p53 bands at the distinct time points shown were quantified and normalized to the zero time points.
Protein loading dye
Manufactured by Sangon
Sourced in China
5× Protein Loading Dye is a concentrated solution used to prepare protein samples for gel electrophoresis. It contains dyes and other components that help visualize and track the movement of proteins during the electrophoresis process.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Proteinsafe protease inhibitor cocktail, by Transgene (2 mentions)
Fatp4, by Proteintech (1 mentions)
β actin, by Proteintech (1 mentions)
Caspase 3, by Proteintech (1 mentions)
Whole protein extraction kit, by Keygen Biotech (1 mentions)
Bca protein quantification kit, by Keygen Biotech (1 mentions)
Agar, by Thermo Fisher Scientific (1 mentions)
Tryptone, by Thermo Fisher Scientific (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Yeast extract, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide lps, by Beyotime (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Ciprofloxacin, by Merck Group (1 mentions)
Vancomycin, by Sangon (1 mentions)
Erythromycin, by Sangon (1 mentions)
Malonate, by Sangon (1 mentions)
Propidium iodide, by Sangon (1 mentions)
Proteinase k solution, by Sangon (1 mentions)
10 protocols using protein loading dye
1
Substrate Ubiquitination Assay for p53
2
Western Blot Analysis of Intestinal Proteins
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The separated tissues of jejunum in mice were homogenized using a Whole Protein Extraction Kit (KeyGen Biotech, Nanjing, P.R. China), and protein concentrations were quantified using a BCA Protein Quantification Kit (KeyGen Biotech, Nanjing, P.R. China). The total protein of IPEC-J2 cells was prepared using Protein Loading Dye (Sangon Biotech, Shanghai, P.R. China).
Equivalent proteins were separated using 15% SDS-PAGE, electro-blotted onto polyvinylidene fluoride membranes, and then blocked with 5% fat-free milk. Then, membranes were incubated overnight at 4 °C with primary antibodies for CD36, FATP4, caspase-3, and β-actin (all purchased from Proteintech Group, Wuhan, P.R. China), and subsequently incubated with goat-anti-rabbit IgG secondary horseradish peroxidase-conjugated antibody (CST, Boston, MA, USA) for 1 h at room temperature. The protein blots were photographed using a Tanon 4200SF Chemiluminescent Imaging System (CliNX, Shanghai, P.R. China). Intensity of blots was quantified using ImageJ software.
Equivalent proteins were separated using 15% SDS-PAGE, electro-blotted onto polyvinylidene fluoride membranes, and then blocked with 5% fat-free milk. Then, membranes were incubated overnight at 4 °C with primary antibodies for CD36, FATP4, caspase-3, and β-actin (all purchased from Proteintech Group, Wuhan, P.R. China), and subsequently incubated with goat-anti-rabbit IgG secondary horseradish peroxidase-conjugated antibody (CST, Boston, MA, USA) for 1 h at room temperature. The protein blots were photographed using a Tanon 4200SF Chemiluminescent Imaging System (CliNX, Shanghai, P.R. China). Intensity of blots was quantified using ImageJ software.
3
Western Blot Analysis of Viral Proteins
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Tissues were homogenized in PBS and centrifuged at 12,000 × g for 20 min to obtain the supernatant. The protein concentration was determined using the Bradford assay. Protein Loading Dye (Sangon, Shanghai, China) was added in the protein solution before boiling 100°C for 5 min and centrifugation at 8,000 × g for 3 min. The protein samples were separated by 12.5% SDS-PAGE with a loading of 100 μg per well. The proteins were transferred onto a nitrocellulose membrane. The membrane was blocked with 3% nonfat milk dissolved in Tris-buffered saline (TBS), incubated with specific antiserum (1:1,000 dilution) for 2 h at 37°C, washed trice with TBST (TBS with 0.05% Tween 20), incubated with horseradish peroxidase (HRP)-labeled secondary antibody (1:10,000 dilution, Zhongshan, Beijing, China) for 1 h at 37°C, and washed trice with TBST. The bands were visualized using the High-sig ECL western blotting substrate (Tanon, Shanghai, China), and detected using a 5200 Chemiluminescence Imaging System (Tanon). The antiserum specific to ccCL, VP28 and β-actin were prepared previously by immunizing New Zealand rabbits with the recombinant proteins.
4
Isogenic E. coli Strain Generation
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Isogenic E. coli K-12 strains (see Table S1 in the supplemental material) were obtained from the Keio collection (52 (link)) or constructed using CRISPR genome editing technology (53 (link)). Plasmids and primers used for this work are listed in Table S4. E. coli strains were cultured by shaking at 200 rpm aerobically at 37°C in Luria-Bertani (LB) broth or by plating on LB agar incubated at 37°C. Yeast extract, tryptone, agar, and 5(6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) were obtained from Thermo Fisher Scientific Corp. (Waltham, MA, USA). Ciprofloxacin, ampicillin, and kanamycin were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Vancomycin, erythromycin, lysine monohydrochloride, arginine hydrochloride, histidine hydrochloride, malonate, propidium iodide, proteinase K solution, 5× protein loading dye, dimethyl sulfoxide (DMSO), and 2.2′-bipyridyl were purchased from Sangon Biotech Inc. (Shanghai, China). Lipopolysaccharide (LPS) was bought from Beyotime Biotech Inc. (Jiangsu, China). Ceftriaxone (Roche, Shanghai, China), and meropenem (Shenghuaxi Pharmaceutical Co., Chongqing, China) were gifts from the Zhongshan Hospital (Xiamen, China) pharmacy.
5
Porcine Granulosa Cell Protein Extraction
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Porcine GCs were lysed in radioimmunoprecipitation assay (RIPA) (Bioworld, Nanjing, China) containing 1% phosphatase inhibitor (v/v). The protein concentration was determined by the BCA Protein Assay Kit (Beyotime, Shanghai, China) and diluted to the same concentration using the 5 × Protein Loading Dye (Sangon, Shanghai, China). Total protein extracts were separated on using SDS-PAGE on 12% gels and electrophoresed for 1 h. Following that, the total protein was transferred onto a PVDF membrane (Millipore, Billerica, MA, USA) and the membrane was blocked with 5% non-fat milk for 2 h. After washing with tris buffered saline tween (TBST) for 15 s, the membrane was incubated with primary antibodies (1:1000 dilution) at 4 °C for 12 h. After that, the membrane was washed thrice for 10 min each time using TBST and incubated with the appropriate secondary antibodies (1:2000 dilution). Chemiluminescence was detected by WesternBrightTM BCL (Advansta, Menlo Park, CA USA).
6
Extraction and Separation of Erythrocyte and Leukocyte Membrane Proteins
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In order to avoid the interference of hemoglobin, erythrocyte ghosts were prepared by incubating the erythrocytes or cryosilicified erythrocytes in 0.25× PBS overnight and collecting by centrifugation (12,000 × g, 10 min), and then the membrane proteins were extracted with the Membrane Protein Extraction Kit (Sangon). The whole proteins of leukocytes and cryosilicified leukocytes were extracted from the samples using the Tissue or Cell Total Protein Extraction Kit (Sangon). All the obtained proteins were dissolved in 5× Protein Loading Dye (Sangon) and then run on 8% SDS-PAGE gel in 1×Tris-Glycine running buffer using Mini-PROTEAN tetra system (BIO-RAD). The samples were run at 120 V for 1.5 h. The polyacrylamide gel was stained in Coomassie Brilliant Blue R250 Protein Stain Reagent for observation.
7
Conditioned Medium Protein Extraction
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Cells cultured at 80% confluence were fed with fresh medium. After 24 h, the supernatant was collected as conditioned medium (CM). The CM was centrifuged using Amicon® Ultra-15 Centrifugal Filters (ufc903096, Millipore, Billerica, MA, USA), and then 5× protein loading dye (Sango Biotech, Shanghai, China) was added to obtain protein lysates for ECM1 detection by western blot.
8
Western Blot Analysis of VEGFA, Tubulin, and CASP3
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GCs were washed with cold PBS and lysed with RIPA buffer containing 1% phosphatase inhibitor (v/v) (Beyotime, Shanghai, China) and proteinase inhibitor (Sigma, St. Louis, MO, USA). The protein concentration was determined with a BCA Protein Assay Kit (Beyotime, Shanghai, China), and samples were diluted to the same concentration using 5× Protein Loading Dye (Sangon, Shanghai, China). Total protein extracts were separated by SDS-PAGE on 12% gels. The proteins were then transferred onto PVDF membranes (Millipore, Billerica, MA, USA), and the membranes were blocked with 5% non-fat milk for 2 h. After washing with Tris-buffered saline with Tween (TBST) for 15 s, the membranes were incubated overnight at 4 °C with anti-VEGFA (diluted 1:5000, ab9570, Abcam, Cambridge, MA, USA), anti-Tubulin (diluted 1:1000, 10094-1-AP ProteinTech, Nanjing, China), and anti-CASP3 (diluted 1:1000, 19677-1-AP, ProteinTech, Nanjing, China). Then, the cells were incubated with a secondary peroxidase-conjugated antibody (diluted 1:2000, Cell Signaling Technology, Beverly, MA, USA) for 1 h at room temperature. Chemiluminescence was detected by WesternBright™ ECL (Advansta, Menlo Park, CA, USA) and analyzed using the ImageJ software (Version 1.51w). Each experiment was performed three times.
9
Immunoprecipitation of sDscam Proteins
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Sf9 cells were infected with recombinant viruses containing HA- or Myc-tagged sDscam and incubated in six-well plates at 27°C for 3 days. Infected cells were washed three times with ice-cold D-PBS, collected by centrifugation at 1000g for 5 min at 4°C, and then homogenized in immunoprecipitation lysis buffer (Thermo Fisher Scientific, 87787) supplemented with 100× phenylmethylsulfonyl fluoride (PMSF; Beyotime, ST505) and 100× ProteinSafe Protease Inhibitor Cocktail (TransGen, DI111-01).
The supernatant was incubated with anti-HA tag rabbit polyclonal antibody (1:50) at 4°C overnight while mixing. After washing Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific, 88802) according to guidelines, the antigen sample/antibody mixture was added to the prewashed magnetic beads and incubated at 4°C for 1 to 3 hours while mixing. Subsequently, the beads were collected using a magnetic stand and washed three times with beads wash buffer. Next, the collected beads were mixed with 80 μl of 5× Protein Loading Dye (Sangon Biotech, C508320-0001) and heated at 96° to 100°C for 10 min. Last, the beads were magnetically separated and the supernatant was stored at −80°C or used for Western blotting.
The supernatant was incubated with anti-HA tag rabbit polyclonal antibody (1:50) at 4°C overnight while mixing. After washing Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific, 88802) according to guidelines, the antigen sample/antibody mixture was added to the prewashed magnetic beads and incubated at 4°C for 1 to 3 hours while mixing. Subsequently, the beads were collected using a magnetic stand and washed three times with beads wash buffer. Next, the collected beads were mixed with 80 μl of 5× Protein Loading Dye (Sangon Biotech, C508320-0001) and heated at 96° to 100°C for 10 min. Last, the beads were magnetically separated and the supernatant was stored at −80°C or used for Western blotting.
10
Protein Expression Quantification from Infected Cells
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For relative expression quantification, infected cells were lysed in radioimmunoprecipitation assay lysis buffer (strong) (Cowin Biosciences, CW2333S) supplemented with 100× PMSF (Beyotime, ST505) and 100× ProteinSafe Protease Inhibitor Cocktail (TransGen, DI111-01). The protein lysate was centrifuged to remove debris. The supernatant was mixed with 5× Protein Loading Dye (Sangon Biotech, C508320-0001) and heated at 96° to 100°C for 10 min. Then, the mixed sample was centrifuged at 13,000 rpm for 20 min at 4°C. The sample was separated by 10% Precast-Glgel Tris-Glycine PAGE (Sangon Biotech, C651101-0001) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, IPVH00010). After probing with the respective antibodies, the PVDF membranes were finally analyzed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34095).
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