Protein of freshly isolated LSECs was purified using triple lysis buffer (TLB: 50mM Tris base, 150mM NaCl, 3mM sodium azide, 12mM sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40) supplemented with 2mM PMSF, 1mM sodium orthovanadate and 1% protease inhibitors cocktail (Santa Cruz sc-24948). 50μg of total protein was used from each sample per lane and resolved on NuPAGE™ Novex™ 10% Bis-Tris Protein Gels. Proteins were transferred onto 0.45μm nitrocellulose membranes via electroblotting using Trans-Blot® SD Semi-Dry (Bio-rad). Membranes were blocked using NAP-BLOCKER™. Blots were probed with primary antibody overnight followed by IRDye secondary antibodies and analyzed using LI-COR Image Studio™. Analyses were normalized to β-actin. All antibodies and dilutions used are listed in Supporting Table S2 .
Protease inhibitors cocktail
Manufactured by Santa Cruz Biotechnology
Protease inhibitors cocktail is a laboratory reagent used to inhibit the activity of proteases, which are enzymes that break down proteins. This product contains a mixture of several protease inhibitors that can effectively prevent the degradation of proteins in biological samples, allowing for the preservation of protein integrity during various experimental procedures.
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Lab products found in correlation
3 protocols using protease inhibitors cocktail
1
LSEC Protein Isolation and Western Blot Analysis
2
Quantification of Colonic Elafin and Cytokines
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Human colonic tissues were homogenized in RIPA buffer with a protease inhibitors cocktail (sc-24948, Santa Cruz Biotechnology). Human sera were diluted ten-fold with reagent diluent and added to the ELISA plates. Elafin was detected with an ELISA kit (DY1747 R&D Systems) as described previously [26 (link)]. Serum cytokines were detected with multiplex ELISA (human 27-plex #m500kcaf0y, Bio-Rad).
3
Hepatic Gene and Protein Quantification
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RNA was prepared from the frozen liver tissue and converted to cDNA using a reverse transcription system (Promega BioSystems, Sunnyvale, CA). Various mRNA transcripts were quantified by qRT-PCR using SYBR-Green in a 7300 real time PCR analyzer (Applied Biosystems, Grand Island, NY). Primer sequences are listed in Table 1 .
Liver protein lysates were prepared in RIPA lysis buffer containing protease inhibitors cocktail (Santa Cruz Biotechnology, Dallas, TX). Proteins were separated by SDS-PAGE and transferred to Immobilon® PVDF membrane (Millipore, Billerica, MA). After immunoblotting with primary Abs [ALR/GFER (Proteintech Group, Inc, Chicago, IL), alcohol dehydrogenases-1 (ADH1), aldehyde dehydrogenases-1 (ALDH1), rabbit polyclonal anti-Bax or anti-Bcl2 (Cell Signaling Technology, Inc., Beverly, MA) washing and incubation with appropriate HRP-conjugated secondary Abs, protein bands were detected using ECL reagent (Thermo Fisher Scientific, Grand Island, NY). β-actin expression was assessed to ensure equal protein loading.
Liver protein lysates were prepared in RIPA lysis buffer containing protease inhibitors cocktail (Santa Cruz Biotechnology, Dallas, TX). Proteins were separated by SDS-PAGE and transferred to Immobilon® PVDF membrane (Millipore, Billerica, MA). After immunoblotting with primary Abs [ALR/GFER (Proteintech Group, Inc, Chicago, IL), alcohol dehydrogenases-1 (ADH1), aldehyde dehydrogenases-1 (ALDH1), rabbit polyclonal anti-Bax or anti-Bcl2 (Cell Signaling Technology, Inc., Beverly, MA) washing and incubation with appropriate HRP-conjugated secondary Abs, protein bands were detected using ECL reagent (Thermo Fisher Scientific, Grand Island, NY). β-actin expression was assessed to ensure equal protein loading.
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