Total protein samples were extracted using the Tissue or Cell Total Protein Extraction Kit (#C510003; Sangon Biotech, China) following the manufacturer’s instructions. The protein concentration was determined using the Modified Bradford reagent (#C100530; Sangon Biotech, China). Approximately 30-μg proteins per cell group were then boiled at 100 °C for five minutes, separated with 10% SDS-PAGE, transferred onto 0.45-μm PVDF membrane (Millipore, USA), blocked with 5% lipid-free milk solution, incubated with primary antibodies overnight at 4 °C, incubated with horseradish peroxide reductase-conjugated secondary antibodies, and finally developed with the enhanced chemiluminescence (ECL) substrates (Thermo Fisher Scientific, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal standard. The primary antibodies used in this study include anti-NLRC4 (#PA5-88997; Invitrogen, USA), anti-PNLRC4 (#MA5-31846; Invitrogen, USA), anti-Caspase1 (#22915-1-AP; Proteintech, USA), anti-Caspase1 p20 (#AG-20B-0042-C100; Adipogen, USA), and anti-GAPDH (#60004-1-lg; Proteintech, USA).
Modified bradford reagent
Manufactured by Sangon
Sourced in China
The Modified Bradford reagent is a laboratory solution used for the quantification of proteins. It is based on the Bradford assay, a colorimetric technique that measures the absorbance shift of the Coomassie Brilliant Blue G-250 dye in response to different protein concentrations.
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Lab products found in correlation
0.45 μm pvdf membrane, by Merck Group (1 mentions)
Tissue or cell total protein extraction kit, by Sangon (1 mentions)
Ag 20b 0042 c100, by Adipogen (1 mentions)
P nitrophenyl β d xylopyranoside pnpx, by Merck Group (1 mentions)
P nitrophenyl β d cellobioside pnpc, by Merck Group (1 mentions)
Whatman, by Cytiva (1 mentions)
2 protocols using modified bradford reagent
1
Protein Extraction and Western Blot Analysis
2
Extracellular Enzyme Activity Assays
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The culture broth was centrifuged at 10,000 rpm, 4 °C for 10 min to collect supernatant. The filter paper enzyme, xylanase, cellobiohydrolase, β-xylosidase, and α-l -arabinofuranosidase activities of culture supernatants were measured using Whatman No. 1 filter paper, beech wood xylan (Yuanye Bio-Technology, China), p-nitrophenyl-β-D-cellobioside (pNPC, Sigma-Aldrich), p-nitrophenyl-β-d -xylopyranoside (pNPX, Sigma-Aldrich), p-nitrophenyl-α-l -arabinofuranoside (pNPA, Sigma-Aldrich) as the substrate respectively, as described previously [26 (link)]. One unit of enzyme activity was defined as the amount of enzyme that liberates 1 μmol of glucose/xylose equivalent or p-nitrophenol from the substrate per minute. The concentration of extracellular proteins was measured using the Modified Bradford reagent (Sangon, Shanghai, China). For SDS-PAGE, equal volumes (24 μl) of culture supernatants were supplemented with 5 × SDS sample loading buffer (GenStar, Beijing, China), boiled for 10 min, and loaded onto a 12 % SDS polyacrylamide separating gel for electrophoresis at 120 V for 1.0–1.5 h.
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