Purification of IgG was described in previous study 22 (link). IgGs from serum samples were isolated using Protein A IgG Purification Kit (Thermo Fisher Scientific). The isolation was manipulated according to the manufacturer's instructions. Briefly, 50μL serum was diluted to 100μL using a proprietary Protein A IgG Binding Buffer. Then the mixture was applied to the protein A plate and washed with 500μL of Binding Buffer three times to remove unbound proteins. Last, the bound IgGs were eluted with 200μL of the proprietary IgG Elution Buffer three times in three separate plates. To determine which fractions contained IgG, the absorbance of each fraction was measured at 280nm by a bicinchoninic acid (BCA) test (Thermo Fisher Scientific). And the fractions containing IgG were stored at -20 °C until the N-glycan release.
Bca test
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BCA (Bicinchoninic Acid) test is a colorimetric assay used to quantify the total protein concentration in a sample. It works by measuring the reduction of copper ions by the protein, which results in a purple-colored complex that can be detected spectrophotometrically.
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Lab products found in correlation
Protein a igg purification kit, by Thermo Fisher Scientific (1 mentions)
Complete mini, by Roche (1 mentions)
Western lightning reagent, by PerkinElmer (1 mentions)
β actin, by Merck Group (1 mentions)
Anti rabbit or anti mouse igg antibodies, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Fusion fx7, by Vilber (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Endoglin, by R&D Systems (1 mentions)
P smad1 5, by Cell Signaling Technology (1 mentions)
Westernbright quantum ecl hrp substrate, by Advansta (1 mentions)
Goat α mouse hrp, by Dianova (1 mentions)
Fluostar omega microplate reader, by BMG Labtech (1 mentions)
Elution buffer, by Thermo Fisher Scientific (1 mentions)
Protein a igg binding buffer, by Thermo Fisher Scientific (1 mentions)
Protein a spin plate for igg screening, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Las 4000, by Cytiva (1 mentions)
8 protocols using bca test
1
IgG Purification from Serum Samples
2
Western Blot Analysis of PrPSc in Brain Tissue
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Western blot analyses were performed as described previously (29 (link)). Briefly, brain tissues were homogenized in 10% (w/v) PBS using microbead-containing tubes and a Ribolyser apparatus (Bio-Rad). Samples were shaken for 45 s and the supernatant was collected through an insulin syringe to obtain a homogeneous suspension. Protein concentrations were measured in each sample using a BCA test (Thermo Fisher Scientific, Illkirch, France) and normalized to have an equivalent level of proteins in each sample before PK digestion test. Fifty microliters of brain homogenates were diluted in 450 μl of PBS with 2% sarcosyl and digested with 20 μg/ml of PK for 1 h at 37°C. The reaction was stopped with 50 μl of Complete Mini (Roche, Switzerland) and 50 μl of each sample were mixed with an equal volume of 2× loading buffer and boiled for 5 min. Thirty microliters were then loaded onto 12% SDS-PAGE precast Criterion gels (Bio-Rad, Marnes-la-Coquette) and analyzed by Western blotting, as described previously (29 (link)). PrPSc was detected with the SAF84 mouse monoclonal antibody, as described previously (24 (link)).
3
Western Blot Analysis of Aggrecan in Mouse Neocortex
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Neocortical brain samples from CamK-OSKM and transgenic control mice were homogenized in 20 mM HEPES pH 7.4, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, and 1% Triton X-100 supplemented with protease and phosphatase inhibitors. The protein concentration of each homogenate was determined by the Bradford method using the BCA test (Thermo Fisher, Waltham, MA, USA). Samples were separated on 10% SDS-PAGE and electrophoretically in the presence at 100 mV for approximately 1 h and then transferred to a nitrocellulose membrane (Schleicher & Schuell GmbH). Membranes were blocked by incubation with 5% semi-fat dried milk in PBS and 0.1% Tween 20 (PBS), and then stained with Ponceau dye (Ponceau 0.3% in TCA 3%) to check transfer efficiency. Membranes were incubated with the appropriate primary antibody (diluted in PBS) overnight at 4 °C. Rabbit anti-Aggrecan (1:5000, Merck, ab1031) and β-actin (1:10000; SIGMA A5441), as loading control, were used as primary antibodies. After three washes, the membrane was incubated with a horseradish peroxidase-anti-rabbit Ig conjugate (DAKO), followed by several washes in PBS-Tween 20. The membrane was then incubated for 1 min in Western Lightning reagents (PerkinElmer Life Sciences). Blots were quantified using the EPSON Perfection 1660 scanner and the Image J software plugin.
4
Western Blot Analysis of Kidney Cells and Tissues
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Protein was extracted from HK-2 cells, which were incubated with TGF-β for 48 h, and homogenized kidney tissues were collected two weeks after UUO induction using RIPA buffer containing a full protease inhibitor cocktail (Thermo Fisher, Rockford, IL, USA). Protein concentrations were determined using the bicinchoninic acid (BCA) test (Thermo Scientific, Rockford, IL, USA), and equivalent amounts of protein extracts were separated using 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis. Proteins were subsequently transferred to a nitrocellulose membrane (Millipore Corporation, Bedford, MA, USA). Next, the membranes were blocked with 5% skim milk containing 2% BSA buffer and incubated with specific primary antibodies overnight at 4 °C (Supplementary Table 1 ). Anti-mouse or anti-rabbit IgG antibodies (Thermo Fisher Scientific, Rockford, IL, USA) were used as secondary antibodies. Protein bands were visualized using an improved chemiluminescence system (Advansta, CA, USA).
5
Western Blot Protocol for Protein Analysis
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Cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100 (v/v), 1% IGEPAL (v/v), 0.1% SDS), and protein concentration was measured with BCA test (Thermo Fisher Scientific). Subsequently, lysates were supplemented with Laemmli buffer [93 (link)], boiled at 95 °C for 10 min prior to separation on 10% SDS–PAGE gels. Proteins were transferred onto nitrocellulose membranes (neoLab Migge Laborbedarf-Vertriebs GmbH, Heidelberg, Germany). Membranes were blocked in 5% w/v BSA (Sigma-Aldrich) in TBST and then incubated with primary antibodies overnight at 4 °C. The next day, membranes were incubated with goat-α-rabbit-HRP, goat-α-mouse-HRP (1:10,000, Dianova, Hamburg, Germany), or mouse-α-goat (1:10,000, Santa Cruz Biotechnology), prior to detection with WesternBright Quantum ECL HRP substrate (advansta, Menlo Park, USA) using a Fusion-FX7 (Vilber Lourmat, Eberhardzell, Germany). Primary antibodies used were Endoglin (AF 1097, R&D systems), Caveolin-1 XP (3267, Cell Signaling Technology), EEA1 (610456, BD Transduction Laboratories), pSMAD1/5 (9516, Cell Signaling Technology), and GAPDH (2118, Cell Signaling Technology). The concentration of primary antibodies was 1:1000 in 3% BSA.
6
Quantifying Mast Cell Activation in Intestine
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Total protein was extracted for jejunum and colonic mucosa and submucosa and concentration was determined with a Bicinchoninic acid assay (BCA) test (Thermofisher, Waltham, MA, USA), as previously described [15 (link)]. To determine the beta-hexosaminidase activity, as a marker of mast cell activation [20 (link),21 (link)], 50 µL of colonic and jejunal protein extract was incubated for 90 min with 100 µL of poly-N-acetylglucosamine dissolved in phosphate buffer at 37 °C, 5% CO2. The reaction was stopped by adding 0.4 M Glycine and absorbance at 405 nm was measured using FLUOstar Omega Microplate Reader (BMG Labtech, Ortenberg, Germany). The activity of beta-hexosaminidase is reported as activity units/µg of protein.
7
IgG Isolation and Purification from Plasma
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Protein A Spin Plate for IgG Screening (Thermo Fisher Scientific, Rockford) was utilized in this study to isolate and purify IgG from blood samples. IgG was purified from each plasma sample as previously described (19 (link)). Briefly, 70 µL of blood plasma was first diluted using Protein A IgG Binding Buffer (Thermo Fisher Scientific, 0.5L). The diluted samples were applied to the Spin plate wells and washed five times by Binding Buffer to thoroughly wash away all unbound non-IgG protein components. Last, the bound IgG was eluted by Elution Buffer (Thermo Fisher Scientific, Rockford, 0.5L) three times into three separate collection plates. In order to determine which fractions contained IgG, the absorbance was measured for each fraction at 280 nm by bicinchoninic acid (BCA) test (Thermo Fisher Scientific, 1L). The purity of eluted IgG was validated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Figure S1
8
Cerebellar Neuron Response to Treatments
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