After transfection, cell samples were collected and total protein was extracted. Proteins were incubated with cold lysis buffer (Solarbio, Beijing, China), centrifuged, concentrated, electrophoresed, and subsequently transferred onto polyvinylidene fluoride membranes. After blocking with phosphate-buffered saline containing 5% non fat milk overnight at 4°C (Wang et al., 2018c), membranes were incubated with antibodies against growth-associated protein 43 (GAP43; rabbit monoclonal, 1:1000; Abcam, Cambridge, UK) and GAPDH (rabbit monoclonal, 1:2500; Abcam) overnight at 4°C. The following day, membranes were treated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, 1:5000; Proteintech, Wuhan, China). An enhanced West Pico ECL Substrate kit (Solarbio) was utilized to perform chemiluminescent detection. GAP43 expression was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to GAPDH.
Cold lysis buffer
Manufactured by Solarbio
Sourced in China
The Cold lysis buffer is a specialized solution used to gently disrupt cells and extract their cellular contents, such as proteins, nucleic acids, and other biomolecules. It is designed to maintain the integrity of the target molecules during the lysis process, making it a valuable tool for various applications in molecular biology and biochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Gap43, by Abcam (1 mentions)
Bca protein quantitation kit, by Solarbio (1 mentions)
Gapdh, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bio spectrum gel imaging system, by Analytik Jena (1 mentions)
Bca protein assay kit, by Yeasen (1 mentions)
Imaging systems, by Bio-Rad (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
6 protocols using cold lysis buffer
1
Western Blot Analysis of GAP43 Expression
2
Western Blot Analysis of Cell Signaling Proteins
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Murine protein samples were lysed in cold lysis buffer (Solarbio, Beijing, China, R0010) and quantified by BCA protein quantitation kit (Solarbio, Beijing, China, PC0020). The proteins were denatured in sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer for 5 min. Then, 30 μg protein sample was added to 12% SDS-PAGE gel for separation and transferred to polyvinylidene difluoride (PVDF) membrane by electrophoresis. The membranes were then blocked with 5% non-fat milk for an hour at room temperature and incubated with primary appropriate antibodies at 4 °C overnight. After washing three times with TBST, the membranes were incubated in secondary antibodies for an hour at room temperature. Enhanced chemiluminescence detection system was used to visualize the immunoreactive bands. Image was used to quantify the protein expression from at least three independent experiments. The following primary antibodies were used in Western blot analysis: p53 (1:2000, Proteintech Group, Wuhan, China, 60282-2-lg), p21 (1:2000, Proteintech Group, Wuhan, China, 28248-1-AP), p16 (1:2500, Abcam, Cambridge, UK, ab211542) and GAPDH (1:1000, Proteintech Group, Wuhan, China, 60004-1-lg).
3
Western Blot Analysis of Tumor Markers
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Cold lysis buffer (Solarbio Life Sciences) was used to homogenize tumor tissues or cell pellets. The homogenate was centrifugated for 30 min at 8000 × g. After BCA quantification, supernatants were separated using SDS-PAGE gel and transferred onto a PVDF membrane. The membrane was incubated with anti-LAPTM4B, anti-ETV1, anti-c-Myc, anti-β-catenin, anti-Wnt3A, anti-Wnt1, anti-CXCL8, and anti-GAPDH antibodies (1:1000), washed with 1x TBST and incubated with HRP-conjugated secondary antibodies (1:2000). Following another wash with 1x TBST, the membrane was incubated with 1 ml chemiluminescent solution. Protein bands were visualized using Tanon imaging system. The sources of all antibodies can be found in Supplementary Table 1 . All full-length western blots can be obtained in the supplementary materials .
4
Western Blot Analysis of Liver Proteins
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Total proteins of liver tissues and HepG2 cells were extracted by cold lysis buffer (Solarbio Science and Technology, Beijing, China) containing 1 mmol/L phenylmethyl sulfonyl fluoride. Then bicinchonininc acid (BCA) protein assay kit (TransGen Biotechnology, Beijing, China) was used to detect the protein concentration. Proteins were dispersed through SDS-PAGE (8–12%) and then transferred to PVDF membranes (Millipore, MA, United States). After blocking nonspecific binding sites with 5% dried skim milk, the membranes were individually incubated with the primary antibodies for 4°C overnight. Then, specific antibody binding was detected by horseradish peroxidase-conjugated secondary antibody for 2 h incubation at room temperature. Protein expression was detected by enhanced chemiluminescence (ECL) method with a Bio-Spectrum Gel Imaging System (UVP, CA, United States). The relative expression of target proteins was normalized to that of β-actin.
5
Western Blot Analysis of Tight Junction, Apoptosis, and Alzheimer's Proteins
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In WB experiments, the nasal mucosa of SD rats and the hippocampus of APP/PS1 transgenic mice were extracted with cold lysis buffer (Solarbio, China) containing 1 mmol/L phenylmethyl sulfonyl fluoride. The total protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit (YEASEN, China). The proteins were separated by SDS-PAGE (8%–12%) and transferred onto polyvinylidene difluoride membranes. After blocking non-specific binding sites with 5% dried skim milk for 2 h, the membranes were incubated with primary antibodies overnight at 4°C. Specific antibody binding was detected by incubating with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Protein expression was detected using the enhanced chemiluminescence method with a Bio-Spectrum Gel Imaging System (UVP, United States). The relative expression of target proteins was normalized to that of GAPDH. ZO-1 (dilution 1:1000), occludin (dilution 1:1000), Bax (dilution 1:1000), Bcl-2 (dilution 1:1000), BACE1 (dilution 1:1000), GAPDH (dilution 1:1000), cleaved caspase 3 (dilution 1:1000), goat anti-mouse IgG HRP (dilution 1:5000), and goat anti-rabbit IgG-HRP (dilution 1:5000) were used.
6
Liver Protein Extraction and Western Blot Analysis
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The liver samples were lysed with cold lysis buffer (Solarbio, Beijing, China), and the samples homogenates were centrifuged at 13,000 rpm for 15 min at 4 °C. The protein concentrations of supernatant were determined with a BCA protein assay kit (Beyotime, Jiangsu, China). The protein samples were mixed with loading buffer in a volume ratio of 4:1 and then boiled for 10 min. Subsequently, the protein samples were separated on 5% SDS-PAGE and transferred to a polyvinylidene fl uoride (PVDF) membrane . The PVDF membranes were blocked for 1.5 h in 5% skim milk and subjected to immunoblotting using primary anti-MAPK antibody overnight at 4 °C. Then, the PVDF membranes were washed three time of 10 min each with TBST and incubated with secondary horseradish peroxidase (HRP)-conjugated antibody for 2 h at room temperature. The blotting of membranes was detected by chemiluminescence (ECL) kits (Millipore, Billerica, MA, USA) and Bio-Rad imaging systems. Relative intensities of protein bands were analyzed by Image J software.
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