Serglycin was isolated from the conditioned medium by anion exchange chromatography. The diethylaminoethyl (DEAE) column was equilibrated at 1 mL/min with running buffer (250 mM NaCl, 20 mM Tris, 10 mM EDTA, pH 7.5) before the addition of medium conditioned by cells and the baseline absorbance was re-established with running buffer. Serglycin was eluted using an eluting buffer (1 M NaCl, 20 mM Tris, 10 mM EDTA, pH 7.5) and concentrated. Serglycin-enriched fractions were subsequently concentrated and analyzed for protein concentration using a Coomassie Blue protein assay (Thermo Scientific, Scoresby, Australia). Glycosaminoglycan concentration was determined using the Dimethylmethylene Blue assay (DMMB) as previously described [41 (link)].
Coomassie blue protein assay
Manufactured by Thermo Fisher Scientific
Sourced in Australia, United States
The Coomassie Blue protein assay is a colorimetric method used to quantify the total protein concentration in a sample. It relies on the binding of Coomassie Brilliant Blue G-250 dye to proteins, resulting in a color change that can be measured spectrophotometrically. The intensity of the blue color is proportional to the amount of protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Hek293t 17 cells, by American Type Culture Collection (2 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
Criterion tgx gel, by Bio-Rad (1 mentions)
Las4000 luminescence image analyser, by Fujifilm (1 mentions)
Anti cd63, by BD (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Lumi light, by Roche (1 mentions)
Linear 25k polyethyleneimine, by Polysciences (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Protein a sepharose, by GE Healthcare (1 mentions)
Dmem f12, by Cytiva (1 mentions)
Protein a conjugated sepharose, by GE Healthcare (1 mentions)
Dmem f12, by Lonza (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Linear polyethylenimine, by Polysciences (1 mentions)
4 protocols using coomassie blue protein assay
1
Serglycin Isolation and Characterization
2
Protein Precipitation and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein from 1 mL of each fraction was precipitated using trichloroacetic acid (20% final concentration; Sigma-Aldrich), and protein concentrations were determined using a Coomassie blue protein assay (Thermo Scientific, Rockford, IL). Precipitated protein of each fraction was dissolved in non-reducing Laemmli sample buffer (Biorad, Hercules, CA). Samples were boiled and 15 µL per fraction was loaded on 4–15% Criterion TGX gels (Biorad), and proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). Blots were incubated with anti-CD63 (1 µg/mL, clone H5C6, Becton Dickinson, Franklin Lakes, NJ) or anti-Apo-B100 (500-fold diluted, clone 2-B4, MP Biomedicals, Santa Ana, CA), washed and incubated with a secondary goat-anti-mouse-antibody coupled to horseradish peroxidase (Dako, Glostrup, Denmark). Protein bands were visualised by incubating the PVDF membranes with a fivefold diluted peroxidase substrate (LumiLight, Roche Diagnostics, Almere, The Netherlands) for 5 min, followed by analysis of luminescence using a LAS4000 luminescence image analyser (Fuji, Valhalla, NY).
3
Recombinant Protein Production in HEK293T
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
mIgp5 and the negative control, mIgp5G, proteins were produced by transient transfection of HEK293T/17 cells (ATCC, Manassas, VA) using linear 25K polyethyleneimine (Polysciences, Warrington, PA) and a light to heavy chain plasmid ratio of 3:2. Cells were cultured for 9 days in DMEM/F12 (Cytiva, Logan, UT) with penicillin-streptomycin (Gibco) and 2% immunoglobulin-depleted fetal bovine serum (Cytiva). Media were changed at 3-day intervals. Transfected cell supernatants were clarified by centrifugation at 4000 x g and the secreted proteins purified by affinity chromatography using protein A-Sepharose (GE Healthcare, Pittsburg, PA) and eluted using 0.1M glycine (pH 3.0). Following overnight dialysis in PBS, products were quantified by Coomassie blue protein assay (Thermo-Pierce, Dallas, TX).
4
Fcp5 Protein Production in HEK293T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fcp5 protein was produced by transient transfection of HEK293T/17 cells (ATCC, Manassas, VA, USA) in 100-mm tissue culture dishes using 6 µg of plasmid DNA with 20 µg of linear polyethylenimine (Polysciences, Warrington, PA, USA) per dish and culturing 9 days in DMEM/F12 (Lonza, Walkersville, MD, USA) with 2% (v/v) immunoglobulin-depleted fetal bovine serum (Thermo-Hyclone, Logan, UT, USA) and penicillin–streptomycin (Lonza), with media changes every 3 days. The collected cell supernatants were clarified by centrifugation at 1,500 × g for 10 min and the secreted Fcp5 purified by affinity chromatography using protein A-conjugated Sepharose (GE Healthcare, Pittsburg, PA, USA) with elution by 0.1 M glycine, pH 3.0, followed by dialysis in PBS and quantitation by Coomassie blue protein assay (Thermo-Pierce, Dallas, TX, USA). The integrity of the purified Fcp5 was documented by SDS-PAGE on native and reduced samples with staining by Coomassie brilliant blue, or by periodic acid Schiff staining for carbohydrate (Thermo-Pierce).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!