We collected fresh leaves of Dendropanax morbifera from Bogil Island, which is located in Wando-gun, Jeollanam-do, South Korea. Unlike the general method for isolating plant vesicles [39 (link),40 (link),41 (link)], we have developed a method for isolating EVs from Dendropanax morbifera leaf to facilitate industrial application. Dendropanax morbifera-derived EVs were isolated by processing the leaves with a mixer grinder plus extractor, passing the resulting crude leaf extract through filter paper, and centrifuging the obtained extract at 10,000× g for 10 min. Then, large debris was removed by filtering the supernatant through a 0.22-μm membrane, and then the filtered EVs were concentrated by centrifuging the sample at 5000× g for 10 min at 4 °C in an Amicon Ultra-4 PL 100 K concentrator (Merck Millipore, Darmstadt, Germany) [5 (link)]. After isolating LEVs, we measured the protein concentration using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), and prepared it by dilution with distilled water to calculate equal concentration of LEVs with and without preservatives. After centrifugation, the protein concentration of EVs was estimated using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).
Pierce bicinchoninic acid bca protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Australia
The Pierce bicinchoninic acid (BCA) protein assay kit is a colorimetric detection and quantitation assay used to measure the total protein concentration in a sample. The kit utilizes the bicinchoninic acid (BCA) reaction, which produces a purple-colored complex that exhibits a strong linear absorbance at 562 nm with increasing protein concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (18 mentions)
Protease inhibitor cocktail, by Roche (10 mentions)
Protease inhibitor cocktail, by Merck Group (9 mentions)
Ripa buffer, by Merck Group (8 mentions)
Phosstop, by Roche (6 mentions)
Bovine serum albumin (bsa), by Merck Group (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Phosphatase inhibitor, by Merck Group (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Phosphatase inhibitor cocktail, by Merck Group (3 mentions)
β tubulin, by Abcam (3 mentions)
Phospho akt ser473, by Cell Signaling Technology (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Nitrocellulose membrane, by Merck Group (3 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (3 mentions)
290 protocols using pierce bicinchoninic acid bca protein assay kit
1
Isolation of Dendropanax morbifera Leaf Extracellular Vesicles
2
Protein Expression Analysis in U2OS and Saos-2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The transfected U2OS and Saos-2 cells were treated with radioimmunoprecipitation assay (RIPA) buffer (Solarbio, China) containing a 1% proteinase inhibitor cocktail. After that, the proteins were extracted and quantified with the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, USA). Subsequently, 10% SDS-PAGE-based electrophoresis was carried out to separate the proteins. Next, they were electronically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA) at 72 V for 2 h. After that, 5% nonfat milk buffer was used to block the membranes at room temperature for 2 h. The anti-LEF1 (1:1,000, ab137872; Abcam, United Kingdom) and anti-GAPDH (1:1,000, ab9485; Abcam, United Kingdom) primary antibodies were then prepared and added to the membranes. The mixture was later incubated overnight at 4°C. Subsequently, the horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (1:1,000, ab6721; Abcam, United Kingdom) was prepared and added, and the mixture was incubated for 1 h at room temperature. The membrane was then washed with phosphate-buffered saline (PBS) three times, and the protein bands were detected using ECL enhanced chemiluminescence Western blotting detection reagents (Beyotime, China). ImageJ software was finally used to calculate the density of the blots.
3
Calcitriol (1,25-(OH)2 vitamin D3) Protocol
4
Radiolabeled Zinc Binding Assay
5
Quantification of Intracellular c-di-GMP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Strains were grown at 28°C under conditions of aeration in LB medium. At the indicated OD578 level, culture samples were harvested, concentrated into 1.5 ml LB, and pelleted again (4°C, 12,700 rpm, 10 min). Sample extraction and analysis of c-di-GMP by liquid chromatography/tandem mass spectrometry (LC-MS/MS) were performed as described previously (43 (link)). Intracellular levels of c-di-GMP were normalized to the corresponding total amount of cellular protein determined using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific). Extractions were performed in biological triplicates.
6
Lipidomic Analysis of Biological Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The internal standard N-pentadecanoyl cerebroside (HexCer(d18:1/15:0)) was obtained from Matreya LLC, Inc. (Pleasant Gap, PA, USA). The internal standard N-palmitoyl 3′-sulfogalactosylceramide ((3′-sulfo)Galβ-Cer(d18:1/16:0)) was purchased from Cayman Chemical, Inc. (Michigan, MI, USA). Internal standards N-heptadecanoyl-D-erythro-sphingosine (d18:1-N17:0 Cer) and N-lauroyl-D-erythro-sphingosylphosphorylcholine (d18:1-N12:0 SM) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). All solvents used for lipid extraction, sample preparation and analysis (including chloroform, methanol, and isopropanol), and a Pierce™ Bicinchoninic Acid (BCA) Protein Assay Kit were obtained from Thermo Fisher Scientific (Pittsburgh, PA, USA). Lithium chloride and lithium hydroxide were purchased from Millipore Sigma Co. (St. Louis, MO, USA). Sample homogenization was performed with Precellys®24 Homogenizer and homogenizing ceramic beads kit (2 mL) from Bertin Instruments (Rockville, MD, USA). N-EVAP nitrogen evaporator (24 positions) was obtained from Organomation Associates, Inc. (Berlin, MA, USA).
7
Optimized LC-MS Proteomics Sample Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Optima® LC–MS grade water, acetonitrile (ACN) and methanol (MeOH) were supplied from Thermo Fisher Scientific (Waltham, MA, USA). Trifluoroacetic acid (TFA) was purchased by Romil Ltd. (Cambridge, UK). Formic acid (FA), dichloromethane (DCM), tris (hydroxymethyl) aminomethane (Tris), pepsin from porcine gastric mucosa (P6887), and pancreatin from porcine pancreas (P7545), α-chymotrypsin from bovine pancreas (CHY5S), TSB (tryptic soy broth) and TBX (tryptone bile X-glucuronide), sodium deoxycholate (SDC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The protease inhibitor cocktail was from Promega (Madison, WI, USA). Ultrapure water (resistivity 18.2 MΩ cm) was obtained by an Arium water purification system (Sartorius, Göttingen, Germany). Solid phase extraction (SPE) C18 cartridges (1 g 6 mL, Agilent Technologies, Santa Clara, CA, USA) were provided by BOND ELUT (Varian, Palo Alto, CA, USA). Cartridges packed with 500 mg Carbograph 4 were supplied from Lara S.R.L. (Lara S.r.l., Formello, Italy). Pierce bicinchoninic acid (BCA) Protein Assay Kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
8
Protein Quantification for Serum and Synovial Fluid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein concentration for days 0 and 84 serum and hock synovial fluid samples (serum diluted 1:100, synovial fluid diluted 1:10 in ultrapure water) was determined for normalization of sample material using the Pierce bicinchoninic acid (BCA) Protein Assay kit (Thermo Fischer Scientific, Waltham, MA) according to the manufacturer’s instructions using bovine serum albumin as the standard. Serum and synovial fluid samples were normalized to 3 mg of protein using 50 mM Tris–HCL buffer (pH 8.0) prior to sample submission.
9
ADSC-SS Starvation and TGF-β1 Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each group’s medium of ADSC-SS was removed and the cells were washed three times using HBSS followed by cell starvation in DMEM low glucose medium which was incubated at 37 °C in 5% CO2 for 24 h. The medium was then aspirated and used for determination of TGF-β1 levels and the total protein concentration secreted by ADSC-SS using ELISA (R&D system, USA) and Pierce™ Bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, USA), respectively, as aforementioned.
10
Western Blot Analysis of CENPF Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were extracted with mammalian protein extraction reagent (M-PER) lysis buffer supplemented with a protease inhibitor and phosphatase inhibitors. The protein concentrations were measured by a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Fisher, Waltham, MA, USA). A total of 30 µg of protein was loaded onto a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and run with 1× SDS running buffer. Proteins were transferred onto a 0.45 µm polyvinylidene fluoride (PVDF) membrane, which was later blocked with 5% milk in tris-buffered saline (TBS)-0.1% Tween-20. Then, the membrane was incubated with anti-CENPF (1:1,000; Proteintech, Rosemont, IL, USA) or anti-β-actin (1:5,000; Proteintech) primary antibody overnight at 4 ℃ and secondary antibody for 1 hour. Finally, proteins were detected using enhanced chemiluminescence (ECL).
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!