Antibody used in western blot for DLL3 was from Proteintech Group (Wuhan, China). Cells were cultured in T-25 or T-75 flasks for 3–5 days at a final confluence of 70%. Cells were collected and lysed with lysis buffer (50 mM Tris–HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 1% Triton X-100, mixed with cocktail proteinase inhibitors from Roche Applied Science (Indianapolis, Indiana)). Protein concentration of the cell lysate was measured by Coomassie Plus (Bradford) Protein Assay from Thermo Scientific (Rockford, Illinois). Fifty micrograms of total protein were run on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for western blot analysis.
Coomassie plus bradford protein assay
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Coomassie Plus (Bradford) Protein Assay is a colorimetric assay used for the quantitative determination of total protein concentration in a sample. The assay utilizes the color change of Coomassie Brilliant Blue dye in response to various concentrations of protein.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase inhibitor cocktail, by Roche (1 mentions)
Chemiluminescent reagent, by Thermo Fisher Scientific (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
40 μm cell strainer, by BD (1 mentions)
Mabselect sure, by GE Healthcare (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Trypsin, by Promega (1 mentions)
5 protocols using coomassie plus bradford protein assay
1
DLL3 Western Blot Protein Quantification
2
Preparation of Apo-bLf and Fe-bLf
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Apo-bLf (iron free) was prepared from commercial grade pure, endotoxin (LPS) free, native bLf. Briefly, 80 mg/ml native bLf was dissolved in Milli-Q water and iron released by reducing pH to 2.06. The bLf solution was then dialysed in 50 kDa molecular weight cut-off dialysis tubing against 0.1 M citric acid for 48 h and pH adjusted back to 8.0. Fe-bLf (iron-saturated) was prepared by the addition of ferric nitriloacetate (Fe-NTA) to Apo-bLf drop wise until the solution reached a deep red colour indication iron saturation. The Fe-bLf solution was then dialysed against Milli-Q water for 48 h. Protein estimation was performed using the Coomassie Plus (Bradford) Protein Assay (Thermo Scientific) and purification was confirmed via SDS-PAGE (results not shown).
3
Investigating NF-kB Activation in Mast Cells
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BMMCs were cultured with rIL-3 and rSCF in the presence or absence of 500 nM TSA and DNP-IgE for 1 h or overnight prior to activation with DNP-BSA. After addition of antigen, cells were incubated for 6–8 h. Whole cell extracts were then obtained using RIPA buffer containing 1% Triton X-100 and quantified with Coomassie Plus (Bradford) Protein Assay (ThermoFisher Scientific). Equal amounts of protein were loaded onto 10% SDS-PAGE gels and transferred to PVDF membrane. Membranes were blocked for 1 h in 5% milk or BSA and incubated overnight with primary antibodies [phospho-relA (1:500) and β-actin (1:5,000)]. Antibodies were obtained from Santa Cruz Biotechnology and Abcam, respectively. Membranes were then washed with PBS tween 20 and incubated with the appropriate secondary antibodies. Membranes were washed once again before the addition of chemiluminescent reagent (Invitrogen). Membranes were imaged using a Biorad Chemidoc.
4
Covalent Coupling of Lysate to PLG Particles
5
Monoclonal Antibody Purification and Characterization
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The mAbs used in this study, identified here as mAb1, mAb2, mAb3, and mAb4, have pIs of 9.4, 9.2, 8.1, and 8.7, respectively. The antibodies mAb1, mAb2, and mAb3 are immunoglobulin (Ig) G1 molecules, while mAb4 is an IgG2. These mAbs were purified at MedImmune (Gaithersburg, MD) and HCP levels were below 20 ppm as measured using custom enzyme-linked immunosorbent assay (ELISA) assay described below. HCPs were obtained from null CHO cells that were grown under similar conditions to mAb producing cells. HCPs in the cell culture fluid were recovered by centrifugation followed by 0.5/0.2 µm filtration. MabSelect SuRe and N-hydroxysuccinimide (NHS)-activated Sepharose 4 Fast Flow resins were obtained from GE Healthcare (Uppsala, Sweden). Coomassie Plus (Bradford) Protein Assay was from Thermo Scientific (Rockford, IL). Materials for SDS-PAGE were from Invitrogen/Life Technologies (Grand Island, NY). Dithiothreitol and iodoacetamide were from Sigma-Aldrich (St. Louis, MO) and trypsin was from Promega (Fitchburg, WI). Hydrophilic polypropylene AcropPrep 96 Filter Plates (0.45 µm) were from Pall Life Sciences (Ann Arbor, MI).
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