Gn truncations (see Fig. S1 in the supplemental material) were cloned into pcDNA3.1 under the cytomegalovirus (CMV) promoter and then transfected into Vero-E6 cells. Lysates were harvested at 48 h in 50 mM dithiothreitol LDS buffer (Thermo Fisher). Samples were heated at 70°C and then loaded into 4 to 12% bis-Tris gels (Thermo Fisher). Proteins were transferred to nitrocellulose membranes using a Mini Blot module wet transfer system (Thermo Fisher). Membranes were blocked in 5% nonfat dry milk (NFDM) in phosphate-buffered saline with 0.1% Tween 20 (PBST) for 1 h and then probed with each anti-Gn MAb, diluted 1:1,000. Bound MAbs were detected using anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Jackson ImmunoResearch), diluted 1:15,000. Membranes were incubated in SuperSignal West Dura extended duration substrate (Thermo Fisher) for 2 min before exposure to CL-XPosure Film (Thermo Fisher) and developed using an SRX-101A film processor (Konica Minolta).
4 to 12 bis tris gels
Manufactured by Thermo Fisher Scientific
4 to 12% bis-tris gels are pre-cast polyacrylamide gel electrophoresis (PAGE) products used for the separation and analysis of proteins. They are designed to provide consistent, high-resolution separation of a wide range of protein sizes.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (2 mentions)
Iblot2 transfer system, by Thermo Fisher Scientific (2 mentions)
Cl xposure film, by Thermo Fisher Scientific (1 mentions)
Srx 101a film processor, by Konica Minolta (1 mentions)
Skim milk, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Novex ecl chemiluminescent substrate reagent kit, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay buffer, by Merck Group (1 mentions)
Prestained protein standards, by Bio-Rad (1 mentions)
Image studio lite version 5, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Traf6, by Cell Signaling Technology (1 mentions)
Il 17ra, by Cell Signaling Technology (1 mentions)
Halt s protease and phosphatase inhibitors, by Thermo Fisher Scientific (1 mentions)
Powerpac 3000, by Bio-Rad (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Iblot system, by Thermo Fisher Scientific (1 mentions)
Mab374, by Merck Group (1 mentions)
Human serum, by Merck Group (1 mentions)
10 protocols using 4 to 12 bis tris gels
1
Gn Truncation Immunoblotting Assay
2
Western Blot Analysis of Protein Extraction
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Total protein was extracted with radioimmunoprecipitation assay buffer (Sigma, Saint Louis, Missouri) containing a protease inhibitor cocktail. After being quantified by a Pierce BCA Protein Assay Kit (Thermo, Waltham, Massachusetts), 20 µg protein was loaded into 4% to 12% Bis-Tris Gels (Thermo, Waltham, Massachusetts) followed by separation. Subsequently, the protein was transferred to polyvinylidene fluoride membrane (Millipore, Germany), and the membrane was blocked with 5% skim milk (Beyotime, China). Thereafter, the membrane was incubated with primary antibodies at 4°C overnight, followed by incubated with secondary antibody at 37°C. After incubation, the membrane was illuminated with NovexECL Chemiluminescent Substrate Reagent Kit (Invitrogen, Carlsbad, California). Finally, the images were taken and quantified by Image J (NIH, Carlsbad, California). Antibodies applied are shown in Table 2 .
3
Quantitative Western Blot Analysis
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SDS-solubilized immune complexes, microcystin-Sepharose bound proteins, and total cell lysates (∼50 μg) were resolved on 4 to 12% Bis-Tris gels (ThermoFisher Scientific) or homemade 10% Tris-glycine gels. Prestained Protein Standards (Bio-Rad) were used as molecular weight standards. Western blotting was performed using the indicated primary antibodies, followed by Infrared IRDye-labeled secondary antibodies and visualization using the Odyssey Infrared imaging system (LI-COR Biosciences). Band intensity was determined using the associated Image Studio Lite version 5.0 Software (LI-COR Biosciences) to accurately quantify protein expression levels. Data were analyzed for statistical significance using GraphPad Prism 9. Differences with p < 0.05 were considered statistically significant.
4
Quantification of Signaling Proteins
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Cells were seeded in 6-well plates as described above for 48 h but at a density of 1 × 106 in 3 mL culture medium. Following this period, the culture medium was replaced with 2 mL fresh culture medium for 2 h. Cells were lysed with radioimmunoprecipitation assay lysis buffer supplemented with Halts protease and phosphatase inhibitors (Thermo Fisher Scientific). Total protein was quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of total cell lysate (30 mg per well) were subjected to sodium dodecyl sulfate electrophoresis on 4 to 12% Bis-Tris gels (Thermo Fisher Scientific). Gels were run at a constant voltage of 100 mV (PowerPac 3000, Bio-Rad) and transferred onto a nitrocellulose membrane with the iBlot2 Transfer System (Thermo Fisher Scientific) as per manufacturer’s instructions. Immunoblotting was completed for GAPDH, IL-17RA, TRAF6 (all Cell Signaling Technology), and IL-17RC (Abcam). A Li-Cor Azure C500 imager was used to visualize immunoblots, and ImageJ software (NIH) was used for quantification.
5
Quantifying Protein Expression by Western Blot
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Protein concentration was determined by bicinchoninic acid assay (Pierce) and then run on 4 to 12% bis-tris gels (Life Technologies). Protein was transferred to a polyvinylidene difluoride membrane using the iBlot system (Thermo Fisher Scientific). Membranes were blocked in 5% milk in tris-buffered saline and Tween 20 (TBS-T) and then probed with primary antibody [Cas9 antibody (7A9-3A3), Santa Cruz Biotechnology, sc-517386, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, Millipore, MAB374] overnight at 4°C. Following washing, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature, and then chemiluminescence was developed with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific).
6
Detecting C3 Binding to Extracellular Vesicles
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For detection of C3 binding to EVs, vesicles were incubated with 20% human serum (Sigma) for 2 h at 37°C. After washing, EVs were resuspended in PBS. The total amount of protein present in purified and serum-treated EVs was quantified using a Pierce bicinchoninic acid (BCA) protein assay kit (Life Technologies, Inc.). Equal amounts of total protein were resolved by SDS-PAGE using 4% to 12% bis-Tris gels (Life Technologies, Inc.) and stained with Coomassie blue. For immunoblotting, proteins on the gels were transferred to polyvinylidene difluoride (PVDF) membranes, blocked with 5% skim milk–PBS–0.1% Tween 20, and incubated with antibodies as indicated. For detection, the following antibodies were used: mouse monoclonal pneumolysin antibody (Abcam, Inc.; final dilution, 1:500); rabbit polyclonal antibodies against GAPDH (1:2,000), LytA (1:2,000) (11 (link)), RrgB (1:1,000), PsaA (1:25,000), and PhtD (1:25,000); and mouse polyclonal antibodies against PspC (1:1,000) and SrtA (1:500). C3 was detected with goat polyclonal anti-C3 (Calbiochem) (1:150). Anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG conjugated to horseradish peroxidase (GE HealthCare) were used as secondary antibodies (1:10,000). Blots were developed with an Amersham ECL Plus Western blotting detection system (GE HealthCare). Pneumolysin in EVs was quantified with ImageJ in respect to purified pneumolysin.
7
Kinase Activity Assay with P70S6K
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Kinase assays were performed as previously described (58 (link)) with minor modifications. Kinase buffer consisted of 25 mM Hepes at pH 7.4 and 50 mM KCl. Just before use, 10 mM MgCl2, 10 nM of the PP1/2 inhibitor calyculin A, 0.5 mM DTT, and 1 μM of the MEK1/2 inhibitor UO126 were added. Karpas-299 cells (2 × 107) in logarithmic phase (0.5 × 106/ml) were lysed in CHAPS lysis buffer. Co-IP was performed using 2 μg of the appropriate antibodies. Captured antibody-protein complexes were preincubated with inhibitors where indicated [FKBP-12/rapamycin (37 μg/ml of FKBP-12 and 10 μM rapamycin), OSI-27 (10 μM), or AZD-8055 (10 μM)] for 30 min at 30°C while shaking. Purified recombinant GST-P70S6KThr389 peptide (80 ng; Millipore) was added to preincubated antibody-protein complexes in the presence of 250 μM ATP and incubated for 30 min at 30°C in a final volume of 30 μl. Kinase reactions were terminated by adding LDS loading buffer to 100 μl, of which 20 μl was loaded on precast 4 to 12% bis-tris gels (Life Technologies). The kinase substrates were visualized using phospho-specific antibodies against p-P70S6KThr389, and a GST antibody (EMD Millipore) was used to control for equal loading.
8
Immunoblotting for Protein Detection
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Confluent plates of cells were harvested, washed with PBS, and lysed with cold RIPA buffer (Sigma Aldrich) with protease inhibitor and phosphatase inhibitor tablets (Sigma Aldrich). Lysates were cleared by centrifugation and protein quantified using the Thermo Fisher BCA Protein Assay protocol. Equal amounts of proteins were prepared in SDS loading buffer supplemented with β-mercaptoethanol, boiled at 95 °C to denature proteins, loaded onto precast 4 to 12% Bis–Tris gels (Life Technologies), and subjected to electrophoresis at 100 V. They were then transferred to PVDF membranes (Life Technologies) with the iBlot2 Transfer System for 7 min or 1h wet transfer at 100V. Membranes were blocked in Intercept Blocking Buffer (LICOR Biosciences) followed by incubation with indicated primary and IRDye-labeled secondary antibodies (LICOR Biosciences). Bands were visualized with the Odyssey® Imaging Systems. Primary antibodies used were as follows: α-Her-2 (Cell Signaling), α-BCAM (R&D), α-α-Tubulin (Abcam), α-Myc-tag (Upstate).
9
Western Blot Protein Extraction and Analysis
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Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM pepstatin A, 1 mM aprotonin, 1 mM leupeptin, 1 mM NaOrthovanadate, and 1% Triton X-100. Protein concentrations were determined by bicinchoninic acid (BCA) assay (Thermo-Fisher Scientific, Loughborough, United Kingdom). Samples were added to 4× gel loading buffer (Life Technologies), 10× dithiothreitol, and boiled at 100°C for 5 min. Fifteen-microgram amounts of samples were resolved on 4 to 12% bis-Tris gels (Life Technologies), transferred to nitrocellulose membranes, and blocked with 5% milk. Membranes were incubated with antibodies (see Table S3 in the supplemental material) and detected by enhanced chemiluminescence (Thermo Scientific).
10
Western Blotting Protocol for Protein Detection
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Western blotting was performed essentially as described [11 (link)]. Briefly, cells were harvested in RIPA lysis buffer containing 1:100 protease inhibitor cocktail (Sigma, Munich, Germany) or PKB lysis buffer [27 (link)] without protease inhibitors. Equal amounts of protein were separated by SDS-PAGE using 4% to 12% Bis-Tris gels (Life Technologies), transferred to nitrocellulose membrane, and incubated over night with anti-TNPO1 antibody (1:1000, ab10303, Abcam), anti-βACTIN (1:100,000, A3853, Sigma) anti-V5 (R960-25, Invitrogen) or anti MYC-antibody (sc40, Santa Cruz Technologies). Next day, membranes were probed with HRP-conjugated secondary antibodies (donkey anti rabbit (sc2305, Santa Cruz Technologies) or goat anti mouse (sc2005, Santa Cruz Technologies), 1:1000 in TBST), and a chemiluminescence assay was performed using Super Signal West Pico substrate (Pierce, Rockford, IL) followed by protein detection.
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