Total protein was extracted from BMDMs by incubating the cells for 10 min at 4 ℃ in RIPA lysis buffer (Fdbio Science, Guangzhou, China) with additional protease inhibitor. After BCA protein determination (Fdbio Science), 5 × loading buffer (Fdbio Science) was added in protein lysates. Equal quantities of lysates were isolated by SDS-PAGE and transferred onto 0.22 um PVDF microporous membranes (Beyotime Institute of Biotechnology, Jiangsu, China). The membranes were blocked with 5% whole milk and then incubated with primary antibodies for 16 h at 4 °C. Afterwards, the membranes were incubated at room temperature for 60 min with the secondary antibodies. Target protein bands were visualized by FDbio-Dura ECL (Fdbio Science). Antibodies applied for western blot in this study were: rabbit anti-IGF2BP3 (Proteintech, Rosemont, USA, 1:1,000, 14642-1-AP), rabbit anti-iNOS (Proteintech, 1:1,000, 22226-1-AP), rabbit anti-CD206 (Proteintech, 1:1,000, 18704-1-AP), rabbit anti-IL-1β (Proteintech, 1:1,000, 16806-1-AP), mouse anti-IL-4 (Proteintech, 1:1,000, 66142-1-Ig), species-matched HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).
Loading buffer
Manufactured by Fdbio
Sourced in China
The 5× loading buffer is a laboratory reagent used to prepare samples for gel electrophoresis. It is a concentrated solution that, when diluted, adjusts the density, pH, and color of the samples to facilitate their loading and visualization on the gel.
Automatically generated - may contain errors
Lab products found in correlation
Dura ecl, by Fdbio (1 mentions)
Rabbit anti igf2bp3, by Proteintech (1 mentions)
Rabbit anti inos, by Proteintech (1 mentions)
Rabbit anti cd206, by Proteintech (1 mentions)
Rabbit anti il 1β, by Proteintech (1 mentions)
Mouse anti il 4, by Proteintech (1 mentions)
Ripa lysis buffer, by Fdbio (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Enhanced chemiluminescence kit, by Fdbio (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Dura ecl kit, by Fdbio (1 mentions)
Phosphatase inhibitors cocktail 2, by Merck Group (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
1 4 dithiothreitol, by Merck Group (1 mentions)
Protein a g bead, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Selleck Chemicals (1 mentions)
Ip lysate buffer, by Beyotime (1 mentions)
6 protocols using loading buffer
1
Western Blot Analysis of Macrophage Markers
2
Protein Extraction and Western Blot Analysis
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For this assay, ccRCC cells or human samples were lysed using RIPA buffer (Beyotime, Shanghai, China) for 15 min on ice and then centrifuged at 12,000 × g for 20 min at 4°C. The supernatants were collected and added to 5× loading buffer (Fdbio, Hangzhou, China). Proteins were further resolved in 10% SDS-PAGE, transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA) and blocked in 5% nonfat milk. The membranes were immunoblotted with appropriate primary and secondary antibodies, and an enhanced chemiluminescence kit (Fdbio) was used to visualize specific protein bands.
3
Protein extraction and western blot analysis
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Cells were lysed and proteins were extracted on ice for 10 min with RIPA lysis buffer (EcoTop, China). Then, the proteins were boiled at 100 °C for 10 min with 5× loading buffer (FDbio, China). Proteins were separated by SDS-PAGE and were then transferred to 0.45 μm PVDF membranes. The membranes were blocked in 5% nonfat milk (for analysis of unphosphorylated proteins) or 5% BSA (for analysis phosphorylated proteins) before they were incubated with primary antibody. The membranes were incubated with secondary antibody the next day. The blots were visualized by an FDBio-Dura ECL Kit (FDbio, China). The antibodies are listed in Additional file 3: Table S3 . The data of western blot was quantitatively analyzed by ImageJ.
4
Immunoprecipitation of Protein Complexes
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Cells were collected and lysed by using IP lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 1 mM EGTA, 5 mM MgCl2, 10%glycerol and 0.2%NP-40), supplemented with protease inhibitor (Thermo Fisher Scientific), phosphatase inhibitors cocktail 2 (Sigma-Aldrich, P5726), and 1 mM 1,4-dithiothreitol (Sigma-Aldrich). Then cell lysate was centrifuged 20000g for 15 min at 4 °C, and the supernatant was incubated with primary antibody for 4 h at 4 °C. After that, the antibody-bound protein was subjected to incubate with Protein A/G Beads (ThermoFisher, 88802) for 2 h at 4 °C. After six washes with wash buffer (0.5 M Tris-HCl pH 7.4, 1.5 M NaCl), immunoprecipitated complexes were denatured with loading buffer (Fdbio science) for 10 min at 95 °C. The samples were then stored at −20 °C or ready for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).
5
Immunoprecipitation and Western Blot Analysis
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NRK-52E cells were lysed with immunoprecipitation (IP) lysate buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing the protease inhibitor (Selleck Chemicals, Texas, United States). After that, the supernatants were collected. Next, the aforementioned cell lysates were incubated with 20 μL protein agarose A/G beads (Thermo, Massachusetts, United States), and the supernatants were obtained by transient centrifugation. Later, an antibody (against CK2α or IgG) was added with shaking for 24 h at 4°C. Subsequently, the aforementioned solutions were incubated with 20 μL protein agarose A/G beads for 4 h at 4°C. Beads were collected by transient centrifugation and washed with IP buffer 3 times. Finally, about 20 μL loading buffer (Fdbio, Hangzhou, China) was mixed with the beads and boiled for 5 min. The method of the Western blot was adopted to detect protein levels.
6
Protein Profiling of Cell Samples and Nanoparticles
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The protein profile of cell lysate, membrane vesicles, PLGA NPs and PLGA@M were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Specifically, samples were prepared at a protein concentration of 2.0 mg/mL in loading buffer (Fdbio, China), and separated by 10% SDS-PAGE, and then stained with coomassie brilliant blue.
For western blot analysis, all samples were mixed with loading buffer to the same total protein concentration of 2 mg/mL, and separated with 10% SDS-PAGE. Then the SDS-PAGE was transferred to a supported nitrocellulose membrane (Pall Life Sciences, Ann Arbor, MI, USA) and blocked with 5% BSA in PBS with 0.1% Tween 20 (PBST). Then, the blots were probed with specific antibodies for rabbit anti-human IL-6 receptor (Abcam, UK), rabbit anti-human IL-1β receptor (Abcam, UK) and rabbit anti-human ACE II (Abcam, UK). Corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies were used to visualize by an enhanced chemiluminescent (ECL) reaction.
For western blot analysis, all samples were mixed with loading buffer to the same total protein concentration of 2 mg/mL, and separated with 10% SDS-PAGE. Then the SDS-PAGE was transferred to a supported nitrocellulose membrane (Pall Life Sciences, Ann Arbor, MI, USA) and blocked with 5% BSA in PBS with 0.1% Tween 20 (PBST). Then, the blots were probed with specific antibodies for rabbit anti-human IL-6 receptor (Abcam, UK), rabbit anti-human IL-1β receptor (Abcam, UK) and rabbit anti-human ACE II (Abcam, UK). Corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies were used to visualize by an enhanced chemiluminescent (ECL) reaction.
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