With the help of protein lysis buffer (Keygen Biotech, Nanjing, China), the extraction of the proteins was conducted from the heart tissues or otherwise the cell samples. BCA reagent kit was utilized to determine the quantity of the protein present in the samples. Then, the protein samples (Lysates) were separated using 10% SDS-PAGE after which it was shifted to polyvinylidene difluoride (PVDF) membranes (Millipore Corp. Billerica, MA, USA). At 4° C overnight, the incubation of the PVDF was performed by HACE1, NRF2, GPX4 and β-actin. Then, for about 2 hours, the horseradish peroxidase-conjugated secondary antibody (Beyotime, 1:5000) was used for the incubation of PVDF. Then, the signal was analyzed by leveraging the chemiluminescence system (Amersham Pharmacia, UK).
Protein lysis buffer
Manufactured by Keygen Biotech
Sourced in China
Protein lysis buffer is a solution used to extract and solubilize proteins from cells or tissues. It disrupts cell membranes and denatures proteins, allowing for the release and isolation of cellular proteins for further analysis or purification.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Beyotime (2 mentions)
Total protein extraction kit prottot 1kt, by Merck Group (2 mentions)
Sds quick match gel kit, by Beyotime (2 mentions)
Flag tag (flag), by Beyotime (1 mentions)
Pvdf membrane, by Abcam (1 mentions)
Ab80425, by Abcam (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
C met, by Proteintech (1 mentions)
Pthrp, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
7 protocols using protein lysis buffer
1
Western Blot Analysis of Protein Targets
2
Western Blot Analysis of Protein Targets
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Proteins were extracted from tissues or cells in protein lysis buffer (Keygen Biotech, Nanjing, China). The proteins were separated by 8%, 10% or 12% SDS–PAGE and then transferred to PVDF membranes (Merck Millipore). The membranes were blocked in 5% skim milk for 2 h and then membranes were incubated overnight at 4 °C with antibodies against Prok2 (Abcam, ab76747, rabbit polyclonal, 1:1000), NeuN (Abcam, ab279296, mouse monoclonal, 1:1000), Acsl4 (Santa Cruz, sc-365230, mouse monoclonal, 1:400), Gpx4 (Santa Cruz, sc-166570, mouse monoclonal, 1:400), Tomm20 (Abcam, ab56783, mouse monoclonal, 1:1000), Tfam (Abcam, ab131607, rabbit polyclonal, 1:1000), MT-ND1(Abcam, ab181848, rabbit monoclonal, 1:1000), GAPDH (YI FEI XUE BIOTECH, YFMA0037, mouse monoclonal, 1:1000), Flag (Beyotime, China, AF519, mouse monoclonal, 1:1000), HA (Beyotime, China, AH158, mouse monoclonal, 1:1000), Fbxo10 (Novus, NBP1-91889, rabbit polyclonal, 1:1000) followed by incubation with horseradish peroxidase-conjugated secondary antibody (Beyotime, China, A0208, A0216, 1:5000) for 2 h. After washing with PBST, protein bands were visualized using SuperSignal® Maximum Sensitivity Substrate (Thermo Fisher Scientific). Samples derive from the same experiment and blots are processed in parallel. Uncropped and unprocessed scans of blots are included in a Source Data file.
3
Comprehensive Protein Expression Analysis
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Total proteins were acquired from the transfected cells with protein lysis buffer (KeyGen, Nanjing, China) and extracted by Total Protein Extraction Kit (PROTTOT-1KT, Sigma-Aldrich). The protein extractions were treated using an SDS Quick Match Gel Kit (P0670, 250 mL, Beyotime Biotechnology, Shanghai, China) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Vicmed, China). The PVDF membranes were subsequently blocked with 5% skimmed milk and incubated with anti-CD44, anti-SOX2, anti-Oct4, anti-Nanog, anti-ZO-1, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-U2AF2, anti-HNRNPC, anti-FUS, anti-DBCRB, anti-DDX54 (ab76947, Abcam), anti-NUCKS1 (ab80425, Abcam), anti-mTOR, anti-SREBP-1c, anti-Cyclin D1, or anti-GAPDH (KC Bio, China) antibodies, respectively, at 4°C overnight. Afterward, their corresponding secondary antibodies were incubated for another hour at room temperature. GAPDH acted as the internal reference.
4
Protein Extraction and Western Blot Analysis
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Proteins were firstly isolated with protein lysis buffer (Keygen, Nanjing, China) and extracted by Total Protein Extraction Kit (PROTTOT-1KT, Sigma-Aldrich, USA). Later, the proteins extracted were treated with SDS Quick Match Gel Kit (P0670-250 ml, Beyotime Biotechnology, Shanghai, China) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Vicmed, China). Afterwards, the membranes blocked by 5% skimmed milk were co-cultivated with the primary antibodies, including Anti-β-actin, Anti-CLDN12and Anti-SRSF1 antibodies, at 4°C overnight. Subsequently, secondary antibodies were added for incubation. Eventually, the expressions of target proteins were analysed by J software. β-actin served as the internal reference.
5
Cytokine and Protease Analysis
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The cells were lysed in Protein Lysis Buffer (KeyGEN) and mixed using an electronic oscillator. After centrifugation at 12,000 rpm and 4 °C for 15 min, the supernatant was collected and stored at − 80 °C. According to the manufacturer’s protocol, the levels of IL-1β and TNF-α in the supernatant were measured using mouse IL-1β and TNF-α ELISA Kits, respectively (Lianke Bio, Hangzhou, China). The level of ADAM8 in patient serum was determined using a Human ADAM8 ELISA Kit (Enzyme-linked Bio, Shanghai, China). We measured the absorbance at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).
6
Western Blot Protein Expression Analysis
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The cells were collected, and total proteins were extracted using protein lysis buffer (KeyGEN BioTECH, Nanjing, China) following the manufacturer’s instructions. Equal amounts of proteins were separated by 10% SDS-PAGE gels using the wet transfer method to transfer the proteins onto PVDF membranes (Millipore, Germany). The membranes were then blocked with 5% skim milk on a shaker at room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4 ∘C on a shaker with the respective primary antibodies, including GAPDH (Proteintech, Rosemont, IL, USA), PTHrP (Proteintech, Rosemont, IL, USA), and c-Met (Proteintech, Rosemont, IL, USA) rabbit monoclonal antibodies. After incubation, the membranes were washed three times with TBS-T for 5 min each to remove unbound primary antibodies. Next, the membranes were incubated with anti-rabbit secondary antibodies (Vector Laboratories, Burlingame, CA, USA) for 2 h at room temperature. Protein expression in each group of cells was visualized using a chemiluminescence imaging system.
7
Testicular Oxidative Stress Pathway Profiling
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Excised testicular tissue, stored at −80 °C, was homogenised by ultrasonic equipment with protein lysis buffer (KeyGEN, Jiangsu, China) and incubated on ice for 10 min. The processed samples were separated by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Polyvinylidene fluoride (PVDF) membranes. Then, the membranes were blocked by 3% bovine serum albumin (BSA) for 1 h at room temperature and probed with primary antibodies (SIRT3, 1:500; NRF2, 1:300; HO-1, 1:800; SOD-1, 1:300; SOD-2, 1:300; AMPK,1:500; and p-AMPK, 1:500) overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibody (1:15,000) for 1 h. Subsequently, the membranes were developed using chemiluminescence reagents.
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