ProteoPrep Blue Albumin and an IgG Depletion kit (PROTBA, Sigma-Aldrich Company, Darmstadt, Germany) were used to deplete serum IgG and albumin. We used 10 µL serums for the depletion, and 1 mL was eluted at the end of the procedure. The Bradford method was used to measure the protein concentration after depletion and vacuum concentration according to the manufacturer’s instructions. Next, 40 µg protein lysate was reduced with 25 mM DTT at 60 °C for 60 min and alkylated with 50 mM iodoacetamide in the dark for 30 min. After alkylation, FASP digestion was performed for each sample using an ultrafiltration filter (10 kDa cutoff, Sartorius, German). Trypsin was added in a 1:100 ratio (enzyme:protein) at 37 °C for 14–16 h, after which the samples were centrifuged at 20,000 ×g (4 °C) for 10 min. The peptides were desalted using Ziptip C18 pipette tips (Merck KGaA, Darmstadt, Germany). After drying, the peptides were resuspended in 0.1% formic acid. Then, 20 µg protein lysate was taken out of each sample and used to build the DIA Spectral Library. The Biognosys’ iRT kit was added to the rest of the samples according to the manufacturer’s instructions (required for DIA analysis using Biognosys’ Spectronaut).
Proteoprep blue albumin
Manufactured by Merck Group
Sourced in Germany
ProteoPrep Blue Albumin is a laboratory reagent used for the detection and quantification of albumin proteins in various sample types. It utilizes a blue-colored dye that binds specifically to albumin, allowing for colorimetric measurement and analysis.
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Lab products found in correlation
2 protocols using proteoprep blue albumin
1
Serum IgG and Albumin Depletion
2
Serum Protein Profiling in Ovarian Cancer
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Serum samples from OVC patients (n = 44) and normal female donors (n = 10) were purchased from Proteogenex (Culver City, CA) and Bioserve (Beltsville, MD). The samples represented diverse tumor stages, age groups and races. 100 μl of serum was used as starting material from all samples and abundant serum proteins were depleted with ProteoPrep Blue Albumin and IgG Depletion Kit (both from Sigma-Aldrich, St. Louis, MO) as described by the manufacturer prior to SDS-PAGE. Twenty micrograms of proteins from eluates in loading buffer (0.5 M Tris–HCl, 0.15 M NaCl, 1% IGEPAL, complete mini [Roche Applied Science, Indianapolis, IN] containing 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM p-methylsulfonylfluoride [PMSF], 1 mM NaVO3, 0.05 M NaF, and 1 mM EGTA) were resolved by SDS-PAGE in 12.5% w/v gels and then further examined by western blot analysis. The membranes were probed with primary anti-KLK6 (H60) and -KLK7 (1407) antibodies (Santa Cruz Biotechnology) and then with appropriate horse radish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). The reactive proteins were visualized by chemiluminescence with SuperSignal West Dura substrate (Thermo Fisher Scientific, Rockford, IL).
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