The L3–L5 DRG tissue was separated and homogenized in RIPA cleavage buffer (P0013, Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (4693132001, Roche, Germany). The protein concentration was determined using a BCA analysis kit (23225, Thermo Fisher Scientific, Waltham, MA, USA). The 30μg protein samples were loaded into the SDS–PAGE gel sample pore and then transferred to the 0.22 μm PVDF membrane. Then, the membrane was blocked with 5% no-fat milk and incubated overnight at 4 °C with antibodies against γH2AX (1:500), H2AX (1:500), H3 (rabbit, 1:200, Cell Signaling Technology, Boston, MA, USA, 7631), ERK (rabbit, 1:1000, Cell Signaling Technology, Boston, MA, USA, 9102S), pERK (rabbit, 1:1000, Cell Signaling Technology, Boston, MA, USA, 9101S), AKT (rabbit, 1:1000, Cell Signaling Technology, Boston, MA, USA, 4691), and pAKT (rabbit, 1:1000, Cell Signaling Technology, Boston, MA, USA, 4060). The images were captured using an Odyssey Imaging System (LI-COR Bioscience, Lincoln, Northeast, Dearborn, MI, USA). ImageJ software was used to analyze the intensity of the selected strip.
Bca analysis kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BCA analysis kit is a colorimetric assay used to quantify protein concentrations in a sample. It is based on the reduction of copper ions by proteins in an alkaline environment, resulting in a purple-colored complex that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey luminescence scanner, by LI COR (2 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Ripa cleavage buffer, by Beyotime (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
P erk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Pakt t308, by Cell Signaling Technology (1 mentions)
P pdk1 ser241, by Cell Signaling Technology (1 mentions)
Ab108327, by Abcam (1 mentions)
Ab48394, by Abcam (1 mentions)
Chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Cadmium chloride cdcl2, by Merck Group (1 mentions)
Anti flag m2, by Cell Signaling Technology (1 mentions)
Anti actin, by Cell Signaling Technology (1 mentions)
6 protocols using bca analysis kit
1
Analyzing DRG Protein Expression
2
Phospho-immunoblotting of Gastric Cancer Cells
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Gastric cancer cells were lysed with Cell Signaling lysis buffer (Cat#40–040, Millipore, Bellerica, USA). Protein concentration was determined via BCA analysis kit (ThermoScientific, Waltham, USA). For phospho-immunoblotting using p-ERK Thr202/Tyr204 (Cat#4376), p-AKT T308 (Cat#9275), p-PDK1 Ser241 (Cat#3438) approximately 50ug of protein was loaded, for total anti-ERK (Cat#9101), AKT (Cat#C67E7), PDK1 (Cat#3062), and actin (Cat#4967, all Cell Signaling, Danvers, USA) 5–10 µg, onto 4–20% SDS/Polyacrylamide gels. Proteins were transferred to nitrocellulose blotting paper via the dry HEP-OWL1 system (ThermoScientific, Waltham, MA). Bands were visualized via the Odyssey luminescence scanner (Li-Cor, Lincoln, USA).
3
SDS-PAGE Western Blot Analysis of Cellular Proteins
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A sodium dodecyl sulfate (SDS) lysate buffer was used to lyse the total protein of cultured cells. The protein concentration was determined using a BCA analysis kit (Thermo Fisher Scientific, USA). Based on whether 8% or 10% SDS polyacrylamide gel electrophoresis was used, the same amount of protein was isolated and transferred to a polyvinylidene difluoride (PVDF) membrane. Reactions with GRP94, caspase‐7, CHOP, TGF‐β, SMAD2, and β‐actin were then performed overnight at 4°C. The PVDF membrane was then washed with PBS‐Tween (PBST) buffer and incubated with Goat anti‐mouse/rabbit IgG (H + L)‐horseradish peroxidase (HRP) (ray antibody biotechnology company, Beijing, China) for one hour. The bands were visualized using an electrochemiluminescence liquid (Merk, Germany), and images were taken using a Tanon 6600 luminous imaging workstation (Tanon, China).
4
Lipophagy Regulation via Protease and Autophagy Inhibitors
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Protease Inhibitor Cocktail (HY-K0010, Med Chem Express), Rapamycin (an activator of lipophagy, Rap, 10219, Med Chem Express) and 3-methyladenine (an inhibitor of lipophagy, 3-MA, 19312, Med Chem Express). BCA Analysis Kit (23225, Thermo Fisher Scientific) and Chemiluminescence reagents (Cat, No.:34094, Thermo Fisher Scientific). Antibodies LC3B (3868s, CST), Actin (4970s, CST), Atg5 (ab108327, Abcam), LC3B (ab48394, Abcam), ATGL (55190-1-AP, Wuhan Sanying) and Lrp1(13213-1-AP, Wuhan Sanying). Cadmium chloride (CdCl2, 202908, Sigma-Aldrich).
5
Flag-tagged Protein Quantification in 293T Cells
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Transfected 293T cells were lysed with Cell Signaling lysis buffer (Cat#40–040, Millipore, Bellerica, USA). Protein concentration was determined via BCA analysis kit (ThermoScientific, Waltham, USA). Approximately 50ug of protein was loaded into a 12% SDS/Polyacrylimide gel. Proteins were transferred to nitrocellulose blotting paper via the dry HEP-OWL1 system (ThermoScientific, Waltham, MA). Nitrocellulose blots were incubated with Anti-Flag M2 (Cat#2368S, Cell Signaling, Danvers, MA) and anti-Actin (Cat#4967, Cell Signalling, Danvers, USA) antibodies. Bands were visualized via the Odyssey luminescence scanner (Li-Cor, Lincoln, USA) according to manufacturer’s instructions.
6
Serum Exosome Isolation and Characterization
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Serum exosomes were isolated using ultracentrifugation as previously described with modifications [14 (link)]. Briefly, the serum was obtained by centrifugation at 3000 × g for 15 min to remove the cell debris and platelets. Microvesicles were pelleted at 10,000 × g for 30 min, after which the exosomes were further purified from the supernatant by ultracentrifugation at 100,000 × g for 60 min. Following isolation, the exosomes were diluted in 100 µL of filtered PBS and stored at − 80 °C.
Exosomes were dissolved in lysis buffer and quantified using a BCA analysis kit (Thermo Fisher Scientific, USA). Western blotting was used to detect exosomal markers, including TSG101, HSP70, CD63 and CD9. Exosomes were fixed in 2.5% glutaraldehyde at 4 °C, dehydrated with gradient alcohol and embedded in epoxy resin. Sections were stained with uranyl acetate and citrate acid lead. The images were captured under a transmission electron microscope (JEM-1010, Japan). For nanoparticle tracking analysis, samples were loaded into the sample chamber of an NS500 unit (NanoSight, UK), and five 1-min videos of each sample were recorded. The data analysis was performed with NTA 2.3 software, and the size and concentration of the particles were calculated.
Exosomes were dissolved in lysis buffer and quantified using a BCA analysis kit (Thermo Fisher Scientific, USA). Western blotting was used to detect exosomal markers, including TSG101, HSP70, CD63 and CD9. Exosomes were fixed in 2.5% glutaraldehyde at 4 °C, dehydrated with gradient alcohol and embedded in epoxy resin. Sections were stained with uranyl acetate and citrate acid lead. The images were captured under a transmission electron microscope (JEM-1010, Japan). For nanoparticle tracking analysis, samples were loaded into the sample chamber of an NS500 unit (NanoSight, UK), and five 1-min videos of each sample were recorded. The data analysis was performed with NTA 2.3 software, and the size and concentration of the particles were calculated.
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