Protein samples were diluted 1:5 with protein loading buffer (6 × Transgen Biotech, Beijing, China) and heated at 95 °C for 5 min. Protein extracts were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride membranes, and blocked with 5% nonfat dry milk in TBST. The membranes were incubated with primary antibodies at 4 °C overnight and with the horseradish peroxidase (HRP)-conjugated secondary antibodies at 37 °C for 1 hour. Protein bands were visualized by enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA) and imaged by a FluorChem M gel documentation system (ProteinSimple, San Jose, CA). The primary antibodies (anti-YAP, anti-p-YAP, anti-LATS1, anti-p-LATS1, anti-MYPT1, anti-p-MYPT1, anti-CREB, anti-p-CREB, anti-GAPDH, anti-α-tubulin, anti-histone H3) and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). CTGF antibody was obtained from Sigma-Aldrich (St Louis, MO, USA).
Protein loading buffer
Manufactured by Transgene
Sourced in China
6× protein loading buffer is a laboratory reagent used to prepare protein samples for electrophoresis. It contains a mixture of chemicals that help denature and solubilize proteins, allowing them to migrate through a gel matrix during the electrophoresis process. The buffer also contains a tracking dye to monitor the progress of the electrophoresis run.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (2 mentions)
Tanon 5200 chemiluminescent imaging system, by Shanghai Tanon Science & Technology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti mypt1, by Cell Signaling Technology (1 mentions)
Anti lats1, by Cell Signaling Technology (1 mentions)
Anti p yap, by Cell Signaling Technology (1 mentions)
Anti p lats1, by Cell Signaling Technology (1 mentions)
Anti p mypt1, by Cell Signaling Technology (1 mentions)
Anti histone h3, by Cell Signaling Technology (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Anti pcreb, by Cell Signaling Technology (1 mentions)
Anti yap, by Cell Signaling Technology (1 mentions)
Anti creb, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by BestBio (1 mentions)
Super ecl detection reagent, by Yeasen (1 mentions)
Protein a g agarose beads, by Merck Group (1 mentions)
Pierce bca assay kit, by Thermo Fisher Scientific (1 mentions)
16 protocols using protein loading buffer
1
Western Blot Analysis of Protein Expression
2
Protein Extraction and Western Blot Analysis
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Protein was extracted from cells or silkworm fat bodies using 1 × RIPA buffer supplemented with 1 × Protease Inhibitor (Bestbio, Shanghai, China). The protein samples were added to 6 × Protein Loading Buffer (Transgen, Beijing, China) and then immediately denatured at 80°C for 10 min. The samples were analysed using 12% SDS-polyacrylamide gels. The proteins were transferred to PVDF membranes (MILLIPORE, Billerica, MA, USA). Immunoblotting was carried out according to standard procedures using Super ECL Detection Reagent (YEASEN, Shanghai, China). The membranes were hybridized with monoclonal anti-ACBP (1:1000 dilution; sc-376853, Santa Cruz), anti-β-Tubulin (1:2000 dilution; TB002-R or TB002-M, Guangzhou Dingguo Biology, China), and anti-LARK (1:1000 dilution) antibodies.
3
Western Blot and Immunoprecipitation Protocol
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Cultured cells were lysed with RIPA buffer with freshly added protease inhibitor cocktail. After incubation on ice, cell lysates were centrifuged and the protein concentration was determined by Pierce BCA Assay Kit (Thermo Fisher Scientific). The supernatants were collected and heated to 95 °C in Protein Loading Buffer (TransGen) for 8 mins. The proteins were resolved by polyacrylamide gel electrophoresis and transferred to PVDF membrane (Millipore). The membranes were then blocked with 5% skim milk in Tris-buffered saline plus 0.1% Tween 20 (TBST) for 1 hour at room temperature and blotted with primary antibodies overnight at 4 °C. After washing the membrane with TBST three times, the membranes were incubated with horseradish peroxidase–conjugated secondary antibodies for 2 hours. The bands were detected using Tanon-5200 Chemiluminescent Imaging System (Tanon Science & Technology).
For detecting RIPK3 and MLKL oligomerization, cells were lysed with non-reducing sample buffer and analyzed by immunoblotting. For immunoprecipitation, cell lysates were centrifuged and the supernatants were incubated with indicated antibodies overnight at 4 °C followed by 5 hours of incubation with 40 μl of Protein A/G agarose beads (Millipore). Beads were washed five times and proteins were eluted by boiling with 2X SDS sample buffer for 8 mins.
For detecting RIPK3 and MLKL oligomerization, cells were lysed with non-reducing sample buffer and analyzed by immunoblotting. For immunoprecipitation, cell lysates were centrifuged and the supernatants were incubated with indicated antibodies overnight at 4 °C followed by 5 hours of incubation with 40 μl of Protein A/G agarose beads (Millipore). Beads were washed five times and proteins were eluted by boiling with 2X SDS sample buffer for 8 mins.
4
Western Blot Analysis of P2Y12, p38 MAPK, and P-p38 MAPK
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The ganglia or cells were homogenized with radio immunoprecipitation assay lysis (Applygen, Beijing, China, Cat# C1053+, 2017). The concentration of protein supernatant was measured by the Lowry method, then diluted with protein loading buffer (Transgen Biotech, China) and bathed in boiling water for 5 min. Aliquots of protein were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene fluoride membrane (Millipore, United States). The membrane was blocked for 2 h, then incubated with rabbit anti-P2Y12 (1:2,000 dilutions, Alomone Labs, Israel), rabbit anti-p38 mitogen-activated protein kinase (p38 MAPK), rabbit anti-phosphorylated-p38 (P-p38 MAPK) (1:1,000 dilutions, Cell Signaling Technology, United States), and mouse monoclonal anti-β-actin (1:800 dilutions, ZSGB-Bio) antibodies at 4°C overnight. After being washed, the membrane was incubated with the secondary antibody (anti-mouse IgG, goat anti-rabbit IgG, 1:2,000, ZSGB-Bio). The Bio-Rad system was used for labeled proteins visualized. Image-J was applied to quantify the results (Zhao et al., 2019 (link)).
5
Isolation and Analysis of C17orf80 Protein Complexes
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After C17orf80-HA or C17orf80-Flag fusion protein was expressed in HeLa cells, the cells were collected and resuspended in a cold mitochondrial isolation solution for mitochondrial isolation as described above. The mitochondria were lysed in NP-40–based lysis buffer (50 mM Tris, 150 mM NaCl, 10% vol/vol glycerol, 1 mM EGTA, 1 mM EDTA, and 0.5% vol/vol NP-40, pH 7.40) at 4°C for 45 min, and the supernatants were collected after centrifugation at 15,000 rpm for 15 min. Precleaned anti-HA magnetic beads (Thermo Fisher Scientific) or anti-Flag magnetic beads (Thermo Fisher Scientific) were added to the supernatants and incubated for 6–8 h at 4°C on a rotator. After washing with TBS (20 mM Tris and 150 mM NaCl, pH 7.4) three times, the beads were boiled in protein loading buffer (TransGen Biotech) for 10 min and then the supernatants were collected for Western blot.
6
Western Blot Analysis of Protein Extracts
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Protein lysates were prepared from cells using RIPA buffer (BioTNT, Shanghai, China) supplemented with a cocktail of protease and phosphatase inhibitors (BioTNT). Protein lysates of EMNVs were prepared using a Total Exosome RNA & Protein Isolation Kit. Lysates were diluted at a ratio of 1:5 with protein loading buffer (6×, Transgen Biotech) and heated at 95 °C for 5 min. Protein extracts were separated on a 10% FastPAGE Plus premixed gel (New Cell & Molecular Biotech, Suzhou, China) at 120 V for 1 h and blotted onto a polyvinylidene difluoride (PVDF) membrane (Merck-Millipore) for 90 min at 200 mA. The membranes were then blocked for 120 min with 5% nonfat milk in TBST (Tris-buffered saline, 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20). Subsequently, the membranes were incubated with primary antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibodies (Cell Signaling Technology, Danvers, MA) at 37 °C for 1 h. The immunoreactive bands were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific) and imaged using an Image Quant LAS 4000 mini bio-molecular imager (GE Healthcare, Uppsala, Sweden).
7
Cross-linking Protein Complexes with DSS
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Disuccinimidyl suberate (DSS) cross-linking was performed as previously described [26 (link)]. The cells were washed 3 times with cold PBS and fully cracked using a 27-gauge needle in 50 µl of ASC reaction buffer (pH 8.0) containing 25 mM Na2PO4, 187.5 mM NaCl, 25 mM HEPES and 125 mM NaHCO3. The mixture was centrifuged at 5000 g for 3 min, and the pellets were cross-linked with 10 µl DSS (25 mM in DMSO) and 40 µl ASC reaction buffer at 37 °C for 30 min. After centrifugation at 5000 g for 10 min, the pellets were dissolved in a solution of 40 µl ASC reaction buffer and 10 µl of 6× protein loading buffer (TransGen, Beijing, China), and the solution was then quenched at room temperature for 15 min, followed by boiling for 10 min. The protein samples were then subjected to western blot analysis using primary antibodies against ASC (Wanleibio, Shenyang, China) at a dilution ratio of 1:500.
8
Western Blot Analysis of Protein Expression
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RIN-m5F cells were cultured in 6-well plates. The growth medium was then discarded and the cells were rinsed three times with PBS. A solution of 2% sodium dodecyl sulfate (SDS) was applied to the cells and the resulting cellular extract was subjected to heat treatment at 100 °C for 10 min then combined with 6× protein loading buffer (J21020, Transgen). The proteins present in the extract were then separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Next, the membranes were blocked with a solution of 5% skim milk powder for 1 h, and then incubated overnight at 4 °C with the appropriate primary antibodies. After three washes with PBST, the membranes were incubated with the appropriate secondary antibodies for 1 h. Finally, the membranes were imaged using an Image Quant LAS4000 Mini system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) after brief incubation with Western-Bright ECL chemiluminescent HRP substrate (32106, SuperSignal West Dura, Thermo Pierce, Waltham, MA, USA).
9
Protein Extraction and Western Blot Analysis
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Proteins were harvested with RIPA buffer (Thermo Fisher Scientific) and denatured in 6 × protein loading buffer (TransGen) and incubated for 10 min at 95°C. Equal samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (10% polyacrylamide) and transferred onto polyvinylidene fluoride (PVDF) membranes by using Mini-PROTEAN Tetra System (Bio-Rad). Membranes were washed three times by TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5% Tween 20) and blocked with 5% non-fat milk in TBST and incubated with the primary antibodies (Calnexin 1:2,000; E 1:1,000) by overnight incubation at 4°C. After washing with TBST three times, membranes were incubated with the secondary HRP-conjugated antibodies and visualized using enhanced chemiluminescence (ECL) system (Bio-Rad).
10
Quantitative Protein Analysis by Western Blotting
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RIN-m5F cells were cultured in 6-well plates. The medium was then removed, and the cells were washed with PBS three times. The cells were treated with 2% sodium dodecyl sulfate (SDS, 200 μL per well). The extract was then heated at 100 °C for 10 min and mixed with 6× protein loading buffer (J21020, Transgen, Beijing, China). The proteins in this extract were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) after the extract was heated for 10 min at 100 °C (4% stacked gel, 15% separated gel, 50 V for 30 min, 90 V for 120 min) and then transferred to nitrocellulose membranes (250 mA, 30–60 min). The membranes were blocked with 5% skimmed milk powder for 1 h and incubated with primary antibody at 4 °C overnight. The membranes were incubated with secondary antibody for 1 h after washing with PBST (PBS plus 0.2% Tween-20) three times. The membranes were then imaged with a luminescent image analyzer (Model: Image Quant LAS4000 Mini, Serial No. 3614294; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) after incubation with WesternBrightTM ECL chemiluminescent HRP substrate (SuperSignal West Dura, 32106, Thermo Pierce) for a few minutes.
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