Proteoextract albumin igg removal kit

All the plasma protein detection and analyses were started from 2016. 40 of 120 samples from PCC patients with typical YDLKS (10), PLC patients with typical YDLKS (10), PCC patients with NS (10) and PLC patients with NS (10) were chosen for characteristic proteins detection using iTRAQ combined with LC-MS/MS technology. Plasma samples were thawed on ice. Equal amounts of plasma from individuals in each group were pooled to yield 2 distinct pools of 500 ml each. To deplete the high-abundance proteins of each plasma pool, a ProteoExtract™ Albumin/IgG Removal Kit (Merck, Germany, Cat 122642) was used according to the manufacture’s instruction. Briefly, plasma was loaded onto the column and proteins bound with high specificity to a bead-based library of diverse peptide ligands. High-abundance proteins which saturated their corresponding ligands were washed out of the column. The remaining low- and mid-abundance proteins in the column were then eluted and collected. After elution, protein was precipitated with acetone overnight at 20°C and dissolved with iTRAQ dissolution buffer. The total protein content of each group was quantified with a Bradford protein assay kit. 10 μg purified samples was taken for SDS-PAGE electrophoresis.

To prepare the cell lysates, cells were extracted in RIPA buffer (PH 8) followed by sonication to disrupt the cellular membranes and centrifugation (10,000× g, 7 min at 4 °C) to remove the cellular debris. A total amount of protein of 2 µg was used to perform the analysis.
Cellular supernatants (1 mL) were centrifuged (1200× g, 10 min at 4 °C) to remove the remaining cells, concentrated an average of 4–5 fold times in Amicon Ultra-4 kgl filter units (Merck Milipore, Burlington, MA, USA) and depleted for albumin/IgGs with the ProteoExtract Albumin/IgG removal kit (Merck Millipore, Burlington, MA, USA).
Sample extracts from the cellular/supernatant fraction were resolved by SDS-PAGE and electro-transferred to nitrocellulose membranes. Detection of the protein of interest was performed with antibodies against total IL-1β (monoclonal anti-IL-1β, Cell Signaling, 1:1000) and cleaved IL-1β (monoclonal anti-IL-1β, Cell Signaling, 1:1000) and normalized with β-actin (monoclonal anti-β-actin, Abcam, dilution:1/5000) or total protein (ponceau). Western blot bands were detected by chemiluminescence using a peroxidase enzymatic reaction (Supersignal, Pierce) and quantified with a ChemiDocTM XRS system using the Quantity-One 1-D analysis software (Bio-Rad).