Analysis with Western blots was conducted to investigate proteins related to JNK and Caspase-3. Cell Total Protein Extraction Kit (Sangon Biotech) was used to extract total cell protein, and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate 20 mL of total protein per well. After electrophoresis, the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane which was soaked in methanol for 2 min and then cleaned once with the membrane transfer liquid. At 4°C, the regulating voltage was set to 90 V and was kept running for 1 h–1.5 h following one and two fight incubations. Finally, the membranes were exposed to X-ray films, and the resultant films were photographed with the X-Omat BT Film (Kodak, NY, USA). Protein quantification was then performed with a Gel-Pro Analyzer 4.0 (Media Cybernetics, WDC, USA), and bands with a positive response were quantified by densitometry and normalized against GAPDH.
Cell total protein extraction kit
Manufactured by Sangon
Sourced in China
The Cell Total Protein Extraction Kit is a laboratory product used for the extraction and purification of total cellular proteins from various cell types. It provides a reliable and efficient method for obtaining a comprehensive protein profile from cells.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Complete protease inhibitor cocktail, by Sangon (2 mentions)
Membrane and cytoplasmic protein extraction kit, by Beyotime (2 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (2 mentions)
X omat bt film, by Kodak (1 mentions)
Gel pro analyzer 4, by Media Cybernetics (1 mentions)
β actin, by Bioworld Technology (1 mentions)
Fibronectin, by BD (1 mentions)
Vimentin, by Proteintech (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
Hdac1, by Proteintech (1 mentions)
Hdac4, by Proteintech (1 mentions)
Snail, by Proteintech (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Canoscan lide 100 scanner, by Canon (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
16 protocols using cell total protein extraction kit
1
Western Blot Analysis of JNK and Caspase-3
2
Comprehensive Protein Extraction and Analysis
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A list of the catalogue and batch numbers of commercially procured reagents is provided in Table S1 . For the whole cell lysate, cells were lysed with a Cell Total Protein Extraction Kit (Sangon Biotech, catalog #C510003) containing a cocktail of phosphatase inhibitors and protease inhibitors. Primary antibodies for AMPK (Proteintech, catalog #10929‐2‐AP), p‐AMPK (Cell signaling, catalog #8208), Fibronectin (BD Transduction Laboratories, catalog #610077), Vimentin (Proteintech, catalog #10366‐1‐AP), N‐cadherin (Abcam, catalog #ab18203), E‐cadherin (Abcam, catalog #ab133597), Snail (Proteintech, catalog #13099‐1‐AP), HK1 (ABclonal, catalog #A1054), HK2 (ABclonal, catalog #A0094), LDHA (ABclonal, catalog #A1146), MCT‐1 (Proteintech, catalog #20139‐1‐AP), G6PI (Proteintech, catalog #15171‐1‐AP), TPI (ABclonal, A2579), HDAC1 (Proteintech, catalog #10197‐1‐AP), HDAC4 (Proteintech, catalog #17449‐1‐AP), HDAC5 (Proteintech, catalog #16166‐1‐AP) and β‐actin (Bioworld, catalog #AP0060) were followed by fluorescently labelled anti‐mouse (Biodragon Immunotech, catalog #BF03001) or anti‐rabbit antibodies (Biodragon Immunotech, catalog #03008), and the blot was then imaged using the Odyssey infrared imaging system (Li‐Cor). Western blots were quantified using ImageJ software. Protein levels were calculated from the ratio of corresponding protein/β‐actin.
3
Spleen Protein Extraction and Western Blot
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Spleen samples were washed twice using prechilled PBS and centrifuged for 5 min at 3000 rpm and 4°C. Supernatant was discarded and pellets were dissolved in RIPA lysis buffer (Sigma, Rolla, Missouri, USA) containing 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma) on ice. Next, the lysate was centrifuged at 12,000 rpm for 10 min at 4°C and supernatant was collected. Cellular protein was extracted by the use of the Cell Total Protein Extraction kit (Sangon Biotech, Shanghai, China). The Pierce bicinchoninic acid protein assay kit (BestBio, Shanghai, China) was used to assess the quality of extracted protein. Western blot was performed as previously described by Han et al. [17 (link)] using rabbit anti-chicken Caspase-3, Caspase-9, β-actin polyclonal (1 : 1500; Abcam, Cambridge, MA, USA), and horseradish peroxidase- (HRP-) labeled IgG secondary antibodies (1 : 800; Abcam). Bands were detected by the use of the enhanced chemiluminescence (ECL) kit (Beyotime, Jiangsu, China) using a CanoScan LiDE 100 scanner (Canon, Tokyo, Japan), and Western blots were analyzed using ImageJ software (v1.80, NIH, USA).
4
Quantitative Protein Analysis in Cells
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Total proteins in cells or tissues were extracted using the Cell Total Protein Extraction Kit (Sangon, China). After separated by SDS-PAGE and transferred to a poly(vinylidene fluoride) (PVDF) membrane, the proteins on membrane were blocked and then incubated with primary antibodies and horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:5000, SA00001-2, Proteintech, China). The primary antibodies were MG53 (1:1000), P16 (1:1000, C610021, Sangon, China), P21 (1:1000, D220091, Sangon, China), P53(1:1000, 10442-1-AP, Proteintech, China), Tau (1:1000, D121297, Sangon, China), p-Tau (Ser396) (1:1000, D155045, Sangon, China), p-Tau (Ser231) (1:1000, D155301, Sangon, China) and p-Tau (Ser235) (1:1000, D155045, Sangon, China), Nrf2 (1:1000, WL02135, Wanlei, China), SOD1 (1:1000, WL01846, Wanlei, China), NQO1 (1:1000, WL04860, Wanlei, China) and Keap-1 (1:1000, WL03285, Wanlei, China), pCNA (1:1000, 10205-2-AP, Proteintech, China), Sirt1 (1:2000, 13161-1-AP, Proteintech, China), β-actin (1:2000, 20536-1-AP, Proteintech, China) and Histone-3 (1:2000, WL0984a, Wanlei, China). β-Actin and Histone-3 were used as internal reference of protein expression in the cytosol and nucleus, respectively.
5
Myocardial Protein Extraction Techniques
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Rat tissue protein was extracted from the myocardium of the peri-infarct zone using a lysis buffer containing 50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS, 1% Na-deoxycholate, 1% protease, (pH 7.5), and complete protease inhibitor cocktail (Sangon Biotech, catalog #C510003). For whole cell lysate, cells were lysed with Cell Total Protein Extraction Kit (Sangon Biotech, catalog #C510003) containing cocktail of phosphatase inhibitors and protease inhibitors. Membrane proteins were extracted using Membrane and Cytoplasmic Protein Extraction Kit (Beyotime, catalog #P0033) and nuclear proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, catalog #P0028) according to the manufacturer’s instructions.
6
Isolation and Quantification of Cardiac Proteins
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The rat heart tissue proteins were extracted from the left ventricular myocardium using a lysis buffer (100 mmol/l Tris-HCl, 4% SDS, 20% glycerine, 200 mmol/l DTT, phosphatase and protease inhibitors, pH 6.8). The cardiomyocytes were lysed using the Cell Total Protein Extraction kit (Sangon Biotech, Shanghai, China) for whole cell lysates. The proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology, Shanghai, China) for nuclear and cytoplasmic lysates according to the manufacturer's protocol. The protein concentrations were measured using a bicinchoninic acid assay.
7
Protein expression analysis by Western blot
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Total protein was extracted from cells using Tissue or Cell Total Protein Extraction Kit (Sangon Biotech). Equivalent protein from different samples was separated by protein electrophoresis, following by transformation onto PVDF membranes (Merck Millipore, Billerica, MA, USA). The membranes were incubated with the anti-rabbit caspase-3, cleaved-caspase-3, caspase-9, cleaved-caspase-9, PINK-1, Parkin, BNIP3, Beclin-1 and LC3A/B (1:1000, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight after immersed into sealed liquid. After the membranes were washed with TBST for several times, goat anti-mouse IgG antibody (1:1000, Cell Signaling Technology) labeled with horseradish peroxidase were incubated with the membranes as a secondary antibody. Anti-mouse β-actin antibody (1:1000, Cell Signaling Technology) was used as a reference protein for normalization. The gray levels of the protein bands were examined by Image J software.
8
Profiling Cellular Metabolic Regulators
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Cells were lysed using the Cell Total Protein Extraction Kit (Sangon Biotech, Shanghai, China) for whole-cell lysate. Total protein was separated by SDS-PAGE and transferred to 0.45 μm polyvinylidene fluoride membranes (MilliporeSigma, Burlington, MA, USA). After blocking with 5% nonfat milk for 1 h, primary antibodies against SLC1A4, SLC1A5, c-MYC, TBX4, TBX15, BACH1, HK2, PFK2, MCT1, GPI, HIF1α, GAPDH and tubulin were separately incubated with the membranes at 4°C overnight. Subsequently, the membranes were incubated with the corresponding secondary antibody for 1 h at room temperature. Eventually, the immunoreacted protein bands were visualised. The antibodies used in this study are listed in supplementary table S4 .
9
Western Blot Analysis of KCNAB2 Protein
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The whole cell lysate was prepared using the Cell Total Protein Extraction Kit (Sangon Biotech, Shanghai, China). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate total protein and the separated protein was then transferred to 0.45 µm polyvinylidene fluoride (PVDF) membranes (MilliporeSigma, Burlington, MA, USA). After blocking with 5% non-fat milk for an hour, primary antibodies against KCNAB2 (17890-1-AP, Proteintech, Wuhan, China) and Tubulin (11224-1-AP, Proteintech, Wuhan, China) were incubated with the membranes individually for an overnight period at 4 °C. Secondary antibodies (ZB-5301, ZSGB-BIO, Beijing, China) were then used for 1 h at room temperature. The immunoreacted protein bands could then be seen. The results from Western blot were analyzed by ImageJ software version 1.8.0.
10
Western Blot Protein Quantification Protocol
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Total protein was extracted from cells or tissues using Tissue or Cell Total Protein Extraction Kit (Sangon Biotech) following the protocol of manufacturer. Equivalent protein (25 µg) from different samples was separated by 10 % SDS-PAGE protein electrophoresis. The separated protein samples were transferred onto the PVDF membranes (Merck Millipore, Billerica, MA, USA). Then, the membranes were incubated with 5 % skim milk to block the non-specific sites. After that, the membranes were incubated with the primary antibodies, anti-rabbit IRF4 or anti-rabbit BCL6 (1:1000, Cell Signaling Technology, Danvers, MA, USA) at 4 °C for 12 h. After the membranes were washed with TBST for several times, the membranes were stained with horseradish peroxidase-conjugated second antibody, goat anti-rabbit IgG antibody (1:1000, Cell Signaling Technology) at room temperature for 1 h. Anti-rabbit β-actin antibody (1:1000, Cell Signaling Technology) was used as a reference protein for normalization. The gray levels of the protein bands were examined by Image J software.
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