Cells were harvested and then resuspended the cells in RIPA buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatases inhibitors (Roche, 4906837001, Mannheim, Germany). The samples were subjected to sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) gels and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, ISEQ00010, Billerica, MA, USA). The membranes were experienced incubation with QuickBlock™ Blocking Buffer (Beyotime, P0228, Shanghai, China), primary antibodies, and IRDye 800CW goat anti-mouse IgG (H + L) or IRDye 680LT donkey anti-rabbit IgG (H + L) (Li-cor, Lincoln, NE, USA) secondary antibody. Immunoreactivity was detected by an Odyssey two-color infrared fluorescence imaging system (Li-cor, Lincoln, NE, USA).
Odyssey two color infrared fluorescence imaging system
Manufactured by LI COR
Sourced in United States
The Odyssey two-color infrared fluorescence imaging system is a lab equipment product by LI-COR. It is designed for the detection and quantification of fluorescently labeled proteins and DNA in a variety of applications, including Western blotting, gel imaging, and microarray analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Irdye 800cw goat anti mouse igg h l, by LI COR (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Irdye 680lt donkey anti rabbit igg h l, by LI COR (2 mentions)
Iseq00010, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail, by Roche (2 mentions)
Bicinchoninic acid bca kit, by Beyotime (2 mentions)
Quickblock blocking buffer, by Beyotime (1 mentions)
Protein assay kit, by Beyotime (1 mentions)
Anti flag m2 bead, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Protease inhibitor mixture, by MedChemExpress (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Bca kit, by Beyotime (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Sds page gel, by Beyotime (1 mentions)
Ripa lysate, by Beyotime (1 mentions)
Protease inhibitor cocktail, by MedChemExpress (1 mentions)
Pvdf membrane, by Affinity Biosciences (1 mentions)
12 protocols using odyssey two color infrared fluorescence imaging system
1
Western Blot Analysis of Protein Expression
2
Whole-cell Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The western blot assay was conducted as previously reported [29 (link)]. In brief, whole-cell lysates were obtained by suspending cells in RIPA buffer (P0013B, Beyotime, Shanghai, China), followed by protein quantification using a BCA (Bicinchoninic Acid) Protein Assay Kit (P0010S, Beyotime, Shanghai, China). Immunoreactivity was visualized by an odyssey two-color infrared fluorescence imaging system (LI-COR Biosciences, NE, USA). β-Tubulin was used as the loading control.
3
Protein-Protein Interaction Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Co-IP and Western blot analysis were performed as described previously (46 (link)). HEK293T cells transfected with the indicated plasmids for 24 h were lysed with cell lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1% Triton X-100, and 10% glycerol) containing 1 mM PMSF (Beyotime) and 1× protease inhibitor mixture (MedChemExpress). The cell lysates were incubated with anti-FLAG (M2) beads (Sigma; catalog no.: A2220-5ML) overnight at 4 °C on a roller. The immunoprecipitants were subjected to electrophoresis. In addition, to identify the interactions between endogenous proteins, PAMs were noninfected or infected with ASFV (1 MOI) for 36 h. The cell lysates then were incubated with an anti-S273R polyclonal antibody or IgG for 8 h at 4 ˚C, and S273R complexes were captured using protein A + G-Sepharose. For Western blotting analysis, equal amounts of cell lysates and immunoprecipitants were resolved by 12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (catalog no.: ISEQ00010; Merck-Millipore). After incubation with primary and secondary antibodies as indicated, the membranes were visualized by an Odyssey two-color infrared fluorescence imaging system (LI-COR).
4
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in ice-cold RIPA lysis buffer (Thermo Fisher Scientific, catalog no. 89901) supplemented with a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Roche, catalog no. 4906845001). The resulting cell lysates were then centrifuged at 12,000 rpm at 4°C for 30 min. Total proteins from the cell extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. Following incubation with the primary and secondary antibodies, the membranes were visualized by enhanced chemiluminescence (ECL) (Thermo Fisher Scientific, catalog no. 32106) or an Odyssey two-color infrared fluorescence imaging system (LI-COR).
5
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples from the cells and animals were lysed in RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor. The proteins were quantified by BCA kits (Beyotime Biotechnology, Beijing). Proteins were separated on a 12% gel and 10% gel transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, United States). The membranes were blocked for 40 min with 5% (w/v) milk dissolved in 0.1% Tween-20 in TBS at room temperature and then were incubated overnight at 4°C with the following primary antibodies: GPX4 (1 : 1000), SCL7A11 (1 : 1000), tublin (1 : 1000, ABclonal, United States, A12289), and GAPDH (1 : 1000). Subsequently, the membranes were washed thrice with TBST, and the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies rabbit/mouse polyclonal antibody for 60 min. Odyssey two-color infrared fluorescence imaging system (LI-COR, United States) was used to visualize the signals. Image-Pro Plus software was used to analyze the relative band densities, and the band densities of target proteins were normalized to that of GAPDH or tublin. All experiments were repeated at least in triplicate.
6
Western Blot Analysis of BMSC Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMSCs were seeded into 6-well plate (2 × 105 cells/well). Cells were lysed using RIPA lysate (Beyotime) and protease inhibitor cocktail (MedChemExpress, Shanghai, China) on the ice. The protein concentration was measured using the BCA kit (BOSTER, Wuhan, China). Each sample (40 µg) was analyzed by 10% SDS-PAGE gel (Beyotime) and transferred to a PVDF membrane (Merck Millipore, Shanghai, China). The membrane was sealed by 5% skim milk at room temperature for 1 h followed by TBST washes thrice for 5 min each. The PVDF membrane was incubated overnight at 4 °C with the following primary antibodies: KDM6B (1:1000, DF13101, Affinity Biosciences, Jiangsu, China), N cadherin (1:1000, AF5239, Affinity Biosciences), CXCR4 (1:1000, AF5279, Affinity Biosciences), GAPDH (1:5000, AF7021, Affinity Biosciences). Next day, the primary antibodies were recycled and membrane was washed with TBST for 3 times for 10 minutes each. The anti-rabbit IgG (H + L) (DyLight™ 680 Conjugate) (1:10000, CST, Shanghai, China) was used as secondary antibody at room temperature for 30 min. After three washes with TBST, the protein expression level was detected by Odyssey two-color infrared fluorescence imaging system (LI-COR, USA) and measured by ImageJ.
7
Immunoblotting Technique for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed immunoblotting as an established protocol84 (link). In brief, the protein samples were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Corporation). The membrane (contained target protein) was blocked in TBST buffer (Beyotime) with 5% nonfat dry milk (2 h, RT) and then incubated with primary antibody (4 °C, overnight). Next, the membrane was washed in TBST several times, and then incubated with secondary antibody (invitrogen). After 2 h, the membranes were washed with TBST several times and were visualized by ECL. The images were taken by an Odyssey two-color infrared fluorescence imaging system (Li-cor, Lincoln, NE, USA).
8
Western Blot Analysis of Exosomal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts from exosomes or liver tissue were separated by 10% SDA-polyacrylamide gel electrophoresis (PAGE) and electrotransferred to a 0.2 µm nitrocellulose membrane. After blocking for 1 h in bovine serum albumin (BSA), the membranes were incubated with primary antibodies at 4 °C overnight and then incubated with a secondary antibody. After the membranes were washed 3 times with TBS and tween 20 (TBST), the protein bands were visualized using an Odyssey two-color infrared fluorescence imaging system (LI-COR, Nebraska, USA) according to the manufacturer’s protocol and analyzed with a gel image analyzer. The antibodies used in this study were as follows: CD63 (ab10895), CD9 (ab92726, Abcam, Cambridge, UK), CD81 (18250-1-AP, Proteintech), NLRP3 (ab214185), ASC (ab47092), caspase-1 (ab1872) (Abcam, Cambridge, UK) and GAPDH (60004-1-Ig, Proteintech, Rosemont, USA).
9
Co-IP and Western Blot Analysis of ASFV Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Co-IP and Western blot analysis were performed, as described previously (49 (link)). At 36 h posttransfection (hpt), the cells transfected with the different plasmids were lysed with the cell lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 5 mM MgCl2, 1 mM EDTA, and 10% glycerol) containing 1 mM PMSF and 1× protease inhibitor mixture (Roche Diagnostics). The cell lysates were incubated with anti-Flag agarose beads (A2220-5ML, Sigma) overnight at 4 °C on a roller. The immunoprecipitants were subjected to electrophoresis. To identify the interactions between endogenous proteins, PAMs were either mock-infected or infected with ASFV (1 MOI) for 36 h. The cell lysates then were incubated with anti-S273R monoclonal antibody or IgG for 8 h at 4 °C, and S273R complexes were captured using protein A+G-Sepharose (Pierce Protein A/G Plus Agarose, Thermo). The equal amounts of cell lysates and immunoprecipitants were resolved by 12% SDS-PAGE and then transferred to polyvinylidine difluoride membranes (ISEQ00010, Merck-Millipore). After incubation with primary and secondary antibodies as indicated, the membranes were visualized by an Odyssey two-color infrared fluorescence imaging system (LI-COR).
10
Western Blot Analysis of PDAC Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PDAC cell lines HPAC and PANC-1 were cultured in culture flasks and collected when they became confluent. Cells were subsequently homogenized in a radioimmunoprecipitation buffer for protein extraction [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, 10 µl/ml protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride; cat. no. P0013B; Beyotime Institute of Biotechnology]. The protein samples were separated by either 8 or 10% SDS-PAGE and subsequently transferred onto nitrocellulose membranes (Merck KGaA). Following 3 washes for 10 min/wash with 20 mM Tris-Cl (pH 7.5), 0.15 M NaCl and 0.05% Tween-20 (TBST), cells were blocked with 5% skimmed milk in TBST for 1 h at room temperature. Membranes were then incubated overnight with primary antibodies at 4°C (Table II ). The membranes were subsequently incubated with secondary goat anti-rabbit IgG HRP-conjugated antibodies (1:1,000; cat. no. 111-625-144; LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature. Protein bands were visualized using a chemiluminescence detection system (Odyssey® two-color infrared fluorescence imaging system; LI-COR Biosciences). Protein levels were normalized to GAPDH (1:10,000; cat. no. G9545; Sigma-Aldrich; Merck KGaA) levels and quantified using ImageJ software version 1.43b (National Institutes of Health, Bethesda, MD, USA).
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!