Immunoprecipitation and Western blotting were performed as described previously [32 (link), 33 (link)]. Briefly, cells were lysed in lysis buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 5 mM sodium orthovanadate, 1% NP-40 and protease inhibitor cocktail (BD Biosciences, San Diego, CA, USA)], and centrifuged at 15,000 rpm for 30 min at 4°C. For immunoprecipitation, equivalent amounts of cell lysates were incubated with the appropriate antibodies followed by incubation with protein A/G agarose beads. Immunoprecipitates were extensively washed and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred, and probed with the proper antibodies. The signal was detected using an enhanced chemiluminescent system (Intron, Seongnam, Korea).
Protease inhibitor cocktail
Manufactured by BD
Sourced in United States
The Protease Inhibitor Cocktail is a solution designed to inhibit the activity of proteases, which are enzymes that break down proteins. This product is used in various laboratory applications to preserve the integrity of protein samples during extraction, purification, and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hybond p membrane, by Merck Group (1 mentions)
Nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Insulin elisa, by Mercodia (1 mentions)
Glu hk m, by Shino-Test (1 mentions)
Sv40 large t antigen, by BD (1 mentions)
Immobilon p polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Pan actin, by BD (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Enhanced chemiluminescence reagent, by PerkinElmer (1 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Antibody against α tubulin, by Merck Group (1 mentions)
Fas cd95, by Proteintech (1 mentions)
Bcl xl, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence detection, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay, by Bio-Rad (1 mentions)
20 protocols using protease inhibitor cocktail
1
Protein Interaction Analysis via IP and Western Blot
2
Western Blot Analysis of Cells and sEVs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and sEVs were lysed in lysis buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 5 mM sodium orthovanadate, 1% NP-40 and protease inhibitor cocktail (BD Biosciences)] and the lysates were centrifuged at 15,000 rpm for 15 min at 4 °C. Equal amounts of the resulting protein extracts were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a Hybond-P membrane (Sigma-Aldrich). The membrane was blocked with 5% skim milk or BSA, and then incubated for overnight with the primary antibody at 4 °C. After washing, the membrane was incubated with the appropriate secondary antibody conjugated to horseradish peroxidase. The signal was detected using the enhanced chemiluminescence system (DoGenBio, Seoul, Republic of Korea). Quantitation was carried out using ImageJ Software (NIH, Bethesda, MD, USA).
3
Immunoprecipitation and Western Blotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoprecipitation and Western blotting were described previously [16 (link)]. Briefly, cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 5 mM sodium ortho vanadate, 1× protease inhibitor cocktail (BD)), and centrifuged at 15,000 rpm for 30 min. For immunoprecipitation, equivalent amounts of cell lysates were incubated with the appropriate antibodies, followed by incubation protein A/G agarose bead. Immunoprecipitates were extensively washed, resolved by SDS-PAGE, transferred to PVDF membrane, and probed with appropriate antibodies. The signals were detected using an enhanced chemiluminescent system (Intron, Seongnam-si, Republic of Korea).
4
Investigating NF-κB Pathway Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Vk2/E6E7 cells were exposed to PICs and NICs and harvested within 60 min followed the treatment with 10–15 min interval. Nuclear and cytoplasmic fractions were obtained as described [53 (link)] with slight modifications. Cells were rinsed twice with ice-cold PBS, scraped, collected by centrifugation at 1000 x g for 5 min, resuspended in STC buffer (0.25 M sucrose, 10 mM tris-HCl, pH 7.5, 3 mM CaCl2) supplemented with protease inhibitor cocktail (BD Bioscience, Bedford, MA). Equal volume of STC buffer, containing 1% Triton X-100 was added to the cell suspension, followed by incubation on ice for 10 min. Nuclear and cytoplasmic fractions were separated by centrifugation at 500 x g for 7 min. Nuclear pellet was resuspended in STC, equal volume of 2 x SDS loading buffer was added to the nuclear and cytoplasmic fractions. Cytoplasmic and nuclear proteins were separated by 10% SDS polyacrylamide gel and transferred to PVDF membrane. IκB-α (degradation in cytoplasm) and NFκB/p 65 (nuclear translocation) were assayed by immunoblotting, as described earlier [34 (link)] using corresponding antibodies, both at 1:1000 dilution. Rabbit anti-human polyclonal antibodies, NFκB/p65 and IκB-α, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)
5
Measuring Serum and Plasma Biomarkers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure serum blood markers such as insulin, triacylglycerol (TAG), and non-esterified fatty acids (NEFAs), samples were allowed to clot for 30 min at room temperature and then centrifuged at 3000 rpm for 10 min. The serum sample was dispensed into plain microtubes and stored at −80 °C until the assay was performed. For plasma glucose measurements, venous blood samples were collected in tubes containing sodium fluoride-ethylenediaminetetraacetic acid. For the measurement of plasma gastric inhibitory polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), venous blood samples were collected in tubes containing a DPP-4 inhibitor and protease inhibitor cocktail (BD, Tokyo, Japan). Thereafter, both samples were immediately centrifuged and stored at −80 °C until analysis. Enzymatic colorimetric assays were used to measure the plasma concentrations of glucose (GLU-HK (M); Shino-test Corporation, Kanagawa, Japan), serum TAG (Pure Auto S TAG-N; Sekisui Medical Company Limited, Tokyo, Japan), and serum NEFAs (Wako Pure Chemical Industries Limited). Enzyme-linked immunosorbent assays were used to measure plasma concentrations of insulin (Mercodia Insulin ELISA; Mercodia AB, Uppsala, Sweden), active GIP, and active GLP-1 (Yanaihara Institute, Inc. ELISA; Yanaihara Institute, Inc. Shizuoka, Japan).
6
Immunoblotting for Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting was performed as previously described [22] (link). Briefly, snap-frozen tissues were homogenized with triton-X/SDS lysis buffer containing 1× Protease Inhibitor Cocktail (BD PharMingen) and phosphatase inhibitors. Samples were resolved by SDS-PAGE and transferred to PVDF membranes overnight at 4°C by electrophoresis. Membranes were blocked at room temperature for at least 1 h using 5% (w/v) milk in 1× TBST. Following overnight incubation with primary antibodies for SV40 large T antigen (1∶250, BD Pharmingen) and actin (CP01, Calbiochem, San Diego, CA), membranes were washed with TBST, incubated with HRP-conjugated secondary antibodies for a minimum of 1 h and protein expression was detected by chemiluminescence.
7
Galectin-3 and MUC1 Protein Analysis
8
Western Blot Protein Extraction and Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells from experimental and control groups were scraped from the 6-well plates and placed into 300 μl of ice-cold lysis buffer [50 mM Tris–HCl (pH∶7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Na-deoxycholate, and 20 μl/ml protease inhibitor cocktail (Pharmingen BD Biosciences, San Jose, CA)]. The samples were clarified by centrifugation at 13,000 rpm for 5 min at 4°C and boiled for 5 min with Laemmli sample buffer containing 100 mM NaF. Protein concentrations were determined by the Bradford method (Bio-Rad Laboratories, Richmond, CA). Equivalent protein amounts were separated on 10% SDS–polyacrylamide gels and transferred to Immobilon-P polyvinylidene fluoride membranes (Millipore Corporation, Bedford, MA). The blots were then hybridized with specific primary antibodies and secondary antibodies labeled with IR Dyes. Signals were observed using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
9
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were lysed using mortar and pestle, resuspended in RIPA buffer (1% Triton, 1% deoxycholate, 0.1% SDS, 0.16M NaCl, 10 mmol/L Tris pH 7.4, and 5 mmol/L EDTA), supplemented with a protease inhibitor cocktail (Pharmingen, San Diego, CA, USA), and analyzed as described previously (48). Primary antibodies used for DEK were as follows: DEK (1:1000; BD Biosciences, San Diego, CA, USA), pan-actin (1:20,000; a gift from James Lessard). Membranes were exposed to enhanced chemiluminescence reagents (Perkin Elmer, Boston, MA, USA) and imaged using the BioRad Chemidoc (Hercules, CA, USA).
10
Essential Oil Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 cells were seeded in a 6-well plate at a density of 2.5 × 105 cells/mL for 24 h and then treated with or without essential oils. After 48 h, the cells were harvested and lysed with ice-cold lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1% NP-40, 5 mM sodium orthovanadate, and protease inhibitor cocktail (BD Biosciences, USA) to extract the total protein. Protein lysates were centrifuged at 22,000 ×g for 10 min at 4°C. Protein concentration in the supernatants was determined using the Bradford method. A total of 20 μg proteins were separated by 12% SDS-PAGE and then transferred to a PVDF membrane (Thermo Fisher Scientific, Inc). The membrane was blocked with 5% nonfat skim milk at room temperature for 1 h, followed by washing with PBS containing 0.1% Tween-20. Thereafter, the membranes were probed with the indicated primary antibodies (Bax, Bcl-2, and caspase-3, 1 : 1,000 dilution) overnight at 4°C. Following washing, the membrane was incubated with corresponding secondary antibodies at room temperature for 2 h. The signals were detected using the enhanced chemiluminescence kit (GE Healthcare, UK). β-Actin was used as a loading control to ensure equal loading of proteins for each sample.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!