To characterize the presence of CD63 and Alix exosomal proteins, exosomes were lysed in RIPA buffer containing 1 mM PMSF and protease inhibitors. The protein concentration of lysates was determined by using the Bicinchoninic Acid Assay (BCA) protein assay kit (Pierce, Waltham, MA, USA). Then, 20 μg of protein were subjected to 12.5% SDS-PAGE under non-reducing conditions for CD63 and under reducing requirements for Alix antibodies. The gel was transferred to a PVDF membrane (MDI) using a wet transfer system (Bio-Rad, Hercules, CA, USA). The blot was blocked in 5% non-fat skimmed milk in 1×TBS-T solution followed by incubation in CD63 primary antibody (1:5000 dilution; Abcam, Cambridge, MA, USA) and Alix primary antibody (1:1000; Santa Cruz, Dallas, TX, USA) overnight at 4 °C. The blot was then washed and incubated with an HRP-conjugated secondary antibody (1:10,000; Invitrogen, Waltham, MA, USA) and developed using an ECL imager (Invitrogen, Waltham, MA, USA).
Ecl imager
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ECL Imager is a laboratory instrument designed for the detection and quantification of chemiluminescent signals. It provides a sensitive and accurate method for analyzing protein samples in Western blot applications.
Automatically generated - may contain errors
Lab products found in correlation
Wet transfer system, by Bio-Rad (3 mentions)
Cd63 primary antibody, by Abcam (2 mentions)
Alix primary antibody, by Santa Cruz Biotechnology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Polyclonal sheep igg anti rabbit cd81, by BioLegend (1 mentions)
Tbst solution, by Abcam (1 mentions)
Ecl reagent kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Hrp conjugated secondary antibody, by Abbkine (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti bcl 2, by BioLegend (1 mentions)
Western breeze chemiluminescent kit, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti mouse igg, by Bio-Rad (1 mentions)
Anti β actin mab, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Bio-Rad (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
9 protocols using ecl imager
1
Exosomal Protein Characterization by Western Blot
2
Exosomal Protein Analysis by Western Blot
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Exosomes (the starting volume was kept constant for both UC and SUC method comparison) were lysed in RIPA buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors. The protein concentration of lysates was measured by using a bicinchoninic acid assay (BCA) protein assay kit (Pierce, part of Thermo Fisher Scientific). Equal volumes of exosomal lysates (50 μL) were subjected to 12.5% SDS-PAGE in non-reducing conditions for CD63 and reducing conditions for Alix and transferred to a polyvinylidene difluoride (PVDF) membrane (MDI Membrane Technologies, Harrisburg, PA, USA) by using a wet transfer system (Bio-Rad Laboratories, Hercules, CA, USA). The blot was blocked in 5% non-fat skimmed milk in 1 × TBS-T solution followed by incubation in CD63 primary antibody (1:5000; Abcam, Cambridge, MA, USA), Alix primary antibody (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (1:3000; Genetix, San Jose, CA, USA) overnight at 4 °C. The blot was washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody and developed by using an ECL imager (Invitrogen, a brand of Thermo Fisher Scientific).
3
Exosomal Protein Expression Analysis
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Exosomes were lysed in RIPA buffer using protease inhibitors and 1 mM phenylmethylsulfonyl fluoride. Exosomal lysates in equal volumes (50 μL) were subjected to non-reducing 12.5% SDS-PAGE for CD63 and CD81 and subsequently transferred utilizing a wet transfer system (Bio-Rad Laboratories, Hercules, CA, USA) on a PVDF membrane (MDI Membrane Technologies, Harrisburg, PA, USA). After blocking, in 1 TBS-T solution (1:5000; Abcam, Cambridge, MA, USA) containing 5 percent nonfat skim milk, the membrane was incubated with the CD63 primary antibody. The antibodies utilized were CD63 (Biolegend, Cat. No. 0353007) and polyclonal sheep IgG anti-rabbit CD81 (Biolegend, Cat. No. 0349509) with antigen affinity purification, as well as beta-actin. They were incubated at 4°C overnight. After being washed, the blot was incubated with a secondary antibody conjugated to horseradish peroxidase (HRP), and it was then developed utilizing an ECL imager (Invitrogen, a brand of Thermo Fisher Scientific).
4
Western Blot Quantification Protocol
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Cells were harvested and lysed in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride and protease inhibitors. The BCA Protein Assay Kit was used to measure the concentrations of total protein. Thereafter, 50 µg of total protein lysate was separated on 10% SDS polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membranes. Next, the membranes were blocked with 5% skim milk in TBST at room temperature for 2 h, incubated overnight at 4 °C with primary antibodies (all 1:1000), followed by incubation with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000). Proteins were visualized using an enhanced chemiluminescence (ECL) imager (Thermo Fisher Scientific), and band intensities were quantified using ImageLab software. β-actin expression was used as control. Three independent experiments were performed for each assay.
5
Protein Isolation and Quantification from Visceral Adipose Tissue
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The total proteins from 100 mg visceral adipose tissue samples were isolated by radioimmunoprecipitation buffer (G-Biosciences, St. Louis, MO, USA) followed by their quantification using a bicinchoninic acid protein assay kit (G-Biosciences, USA). In brief, 30 µg denatured protein was resolved on 10% Tris–glycine gel and transferred to a polyvinylidene fluoride membrane (Bio-Rad, USA). The non-specific binding sites on the membrane were blocked with 5% skimmed milk. The membrane was incubated with corresponding primary antibodies (Abbkine, USA) at 1:700 dilutions overnight at 4°C, and after washing the membrane was treated with an HRP-conjugated secondary antibody (Abbkine, USA) for 2 h. The detection of signals was recorded by the use of an ECL Reagent Kit (Thermo Fisher Scientific, USA) in the gel imaging system (Thermo Fisher Scientific ECL Imager). The protein band density was measured and compared using the ImageJ software.
6
Western Blot Analysis of Cellular Proteins
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Isolated undifferentiated or differentiated colonies (each comprised of ~1 x 103 cells) were immediately placed into sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS w/v, 1mM ü-mercaptoethanol, 10% glycerol)[27 (link)]. Samples were placed into a 95 degrees heat block for 5 min and then loaded onto 4-20% gradient polyacrylamide gels (BioRad Inc. Cat #4561094) with molecular weight markers (Biorad Inc Cat #1610374) and separated by SDS-PAGE. Proteins were transferred to PVDF membrane and blocked with blocking reagent (5% dry milk [Bio Rad Cat #170-6404] in PBS, pH 7.4, with 0.1% Tween 20) at room temperature. The blots were challenged with primary antibody at dilutions of 1/500-1/1000 in block overnight at 4 °C, followed by washing 3 times with PBST (1x PBS + 0.1% Tween 20) at room temperature and challenge with appropriate secondary antibody (1/5,000-1/10,000) conjugated to horseradish peroxidase (Jackson Laboratories) for 2 hours followed by washing 3 times with 1X PBST at room temperature. The blot was then visualized under chemiluminescence in an ECL Imager (Thermo Scientific) using Clarity Western ECL Substrate (Bio Rad Cat #170- 5061). Primary Antibody- Desmin (Rabbit) (Cat #5332P, Cell Signaling). Secondary Antibody- Peroxidase Affinipure Donkey Anti-Rabbit (Cat #711-035-152, Jackson Immunoresearch Labs)
7
Protein Expression Analysis by Western Blot
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Cells were harvested and lysed in RIPA lysis buffer (Beyotime, Jiangsu, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitors. The Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) was used to measure the concentrations of total protein. Equal amount of total protein lysate was separated on 10% SDS-polyacrylamide gel and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Next the membranes were blocked with 5% skim milk in TBST at room temperature for 2 h, incubated overnight at 4 ℃ with primary antibodies (all 1:1000), and followed by incubation with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:2000). Proteins were visualized using an ECL imager (Thermo Fisher Scientific, USA) and band intensities were quantified using ImageLab software. The expression of GAPDH was used as a control. Three independent experiments were performed for each assay.
8
Western Blot Analysis of Drug Resistance
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Total cell lysates (20 μg) from drug-sensitive and -resistant cells were resolved on 6% SDS-PAGE and transferred onto PVDF membrane. The membrane was blocked in milk in phosphate buffered saline (PBS) at 5% (w/v) and probed with P-gp specific mAb (0.1 μg/ml of C219 mAb) or anti-Bcl-2 (0.5 μg/ml; BioLegend, San Diego, USA; anti-Bcl-2 (D55G8) rabbit mAb, 1:1000 v/v (human specific), Cell Signaling Technology, Massachusetts, USA; or anti-β-Actin mAb, 0.5 μg/ml, Sigma-Aldrich, Toronto, CA) in 5% milk/PBS, followed by several washes in PBS and incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000 v/v, BioRad, Ont., CA). The reactive signals were visualized using Western Breeze Chemiluminescent Kit and captured using ECL-imager from Thermo-Fisher Scientific. Tubulin expression was detected on the same PVDF membrane using anti-α-tubulin specific monoclonal antibody (1 μg/ml Sigma-Aldrich, Ont., CA), followed by HRP-conjugated goat anti-mouse IgG (1:5000 (v/v), BioRad, Ont., CA).
9
PCR Fragment Separation and Sequencing
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DNA fragments obtained from PCR were resolved on a 1.5% agarose gel at 100V for 80 min (Bio-Rad, Hercules, USA). DNA bands were visualized using an ECL imager (Thermo Fisher Scientific, Waltham, USA).
For sequencing, DNA samples obtained from PCR were purified using QIAquick PCR purification kit (Qiagen, Venlo, Netherlands). Single pass DNA sequencing was performed by Axil Scientific (1st BASE, Singapore, Singapore).
For sequencing, DNA samples obtained from PCR were purified using QIAquick PCR purification kit (Qiagen, Venlo, Netherlands). Single pass DNA sequencing was performed by Axil Scientific (1st BASE, Singapore, Singapore).
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