Cells were solubilized with lysis buffer [50 mM Tris-HCl (pH7.5), 150 mM NaCl, 5 mM EDTA, 0.5% (v/v) NP-40, 50 mM NaF, 1 mM Na3VO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.25 mM pAPMSF] and centrifuged at 12,000 × g for 20 min. For co-immunoprecipitation assay, cells were sonicated briefly in lysis buffer before centrifugation. The lysates were subjected to immunoprecipitation, SDS-PAGE, and Western blot analyses as described2 (link).
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Western blot
Western blot
Western blot is an analytical technique used to detect and quantify specific proteins in a complex mixture extracted from cells or tissues.
It involves separating proteins by size using gel electrophoresis, transferring them to a membrane, and detecting the protein of interest using specific antibodies.
PubCompare.ai is an innovative platform that can help optimize Western blot protocols by allowing users to easily locate and compare published methods, ensuring the best approach for their experiments.
Leveraging advanced machine learning, PubCompare.ai provides data-driven insights to help achieve consistent, high-quality results and take the guesswork out of the Western blot workflow.
This solution can help researchers streamline their Western blot procedures and improve the reproducibility of their findings.
It involves separating proteins by size using gel electrophoresis, transferring them to a membrane, and detecting the protein of interest using specific antibodies.
PubCompare.ai is an innovative platform that can help optimize Western blot protocols by allowing users to easily locate and compare published methods, ensuring the best approach for their experiments.
Leveraging advanced machine learning, PubCompare.ai provides data-driven insights to help achieve consistent, high-quality results and take the guesswork out of the Western blot workflow.
This solution can help researchers streamline their Western blot procedures and improve the reproducibility of their findings.
Most cited protocols related to «Western blot»
Aprotinin
Biological Assay
Buffers
Cells
Centrifugation
Co-Immunoprecipitation
Edetic Acid
Immunoprecipitation
leupeptin
Nonidet P-40
SDS-PAGE
Sodium Chloride
Tromethamine
Western Blot
Cells were incubated for 24 h in complete media supplemented with 1 µg/ml doxycycline and 50 µM biotin. After three PBS washes, cells (for small-scale analysis, <107; for large scale analysis, 4 × 107) were lysed at 25°C in 1 ml lysis buffer (50 mM Tris, pH 7.4, 500 mM NaCl, 0.4% SDS, 5 mM EDTA, 1 mM DTT, and 1x Complete protease inhibitor [Roche]) and sonicated. Triton X-100 was added to 2% final concentration. After further sonication, an equal volume of 4°C 50 mM Tris (pH 7.4) was added before additional sonication (subsequent steps at 4°C) and centrifugation at 16,000 relative centrifugal force. Supernatants were incubated with 600 µl Dynabeads (MyOne Steptavadin C1; Invitrogen) overnight. Beads were collected and washed twice for 8 min at 25°C (all subsequent steps at 25°C) in 1 ml wash buffer 1 (2% SDS in dH2O). This was repeated once with wash buffer 2 (0.1% deoxycholate, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, and 50 mM Hepes, pH 7.5), once with wash buffer 3 (250 mM LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA, and 10 mM Tris, pH 8.1) and twice with wash buffer 4 (50 mM Tris, pH 7.4, and 50 mM NaCl). 10% of the sample was reserved for Western blot analysis. Bound proteins were removed from the magnetic beads with 50 µl of Laemmli SDS-sample buffer saturated with biotin at 98°C. For the larger scale preparation, 90% of the sample to be analyzed by mass spectrometry was washed twice in 50 mM NH4HCO3.
Biotin
Buffers
Cells
Centrifugation
Deoxycholate
Doxycycline
Edetic Acid
HEPES
Laemmli buffer
Mass Spectrometry
Nonidet P-40
Protease Inhibitors
Proteins
Sodium Chloride
Triton X-100
Tromethamine
Western Blot
SaOS2 cells were transfected with the respective siRNAs and then with the respective expression plasmids for VSVG-GFP, VSVG-Myc, VSVG-MT1-MMP, or VSVG-KDELR-Myc with or without expression plasmid for sr-IFT20 or IFT20. To examine the ER-to-cell surface transport, VSVG-GFP-transfected SaOS2 cells were incubated overnight at 40 °C in DMEM containing 1% FBS to accumulate VSVG-GFP at the ER, and then shifted to DMEM containing 1% FBS and 0.1 mg/ml cycloheximide pre-warmed at 32 °C to allow transport through the Golgi. After 30 or 60 min in culture, cell surface proteins were biotinylated with 0.5 mg/ml Sulfo-NHS-ss-biotin (Thermo) in Dulbecco’s PBS (DPBS) for 30 min at 4 °C and quenched with 50 mM NH4Cl in DPBS for 10 min at 4 °C. After washing with ice-cold DPBS, cells were solubilized with Triton X-100 lysis buffer [25 mM Tris-HCl (pH7.5), 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.4% (w/v) sodium deoxycholate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.25 mM pAPMSF]. Biotinylated proteins were affinity-purified with streptavidin-Sepharose beads and subjected to SDS-PAGE followed Western blot analyses. ER-to-cis-Golgi and intra-Golgi transport of VSVG and VSVG-MT1-MMP and retrograde transport of VSVG-KDELR have been described previously48 (link).
Aprotinin
Buffers
Cells
Cell Surface Proteins
Cold Temperature
Cycloheximide
Deoxycholic Acid, Monosodium Salt
Edetic Acid
Golgi Apparatus
leupeptin
Plasmids
Proteins
RNA, Small Interfering
SDS-PAGE
Sodium Chloride
streptavidin-agarose
sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate
Triton X-100
Tromethamine
Western Blot
Protocol full text hidden due to copyright restrictions
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Actins
Animals
Antibodies
Biopharmaceuticals
Capsule
Catabolism
Cataract
Cells
DNA Replication
Epithelium
Epitopes
Eye
Fibrosis
Fluorescent Antibody Technique
Fluorescent Dyes
Freezing
Gene Expression
Genes
Homo sapiens
Immunoglobulins
Institutional Animal Care and Use Committees
Lens, Crystalline
Mice, Inbred C57BL
Microscopy, Confocal
Mus
Operative Surgical Procedures
Protein Denaturation
Proteins
RNA-Seq
Smooth Muscles
Tissues
Training Programs
Vision
Western Blotting
Seven millilitres of SARS-CoV-2 pseudoviruses with a titre of 1.86 × 105 TCID50/ml were pelleted through a 25% sucrose cushion by ultra-centrifugation at 100,000× g for 3 h. The layers of supernatant and sucrose were removed, and the resulting viral pellets were re-suspended in 100 μl PBS. Sixty microlitre prepared pseudoviruses were mixed with 15 μl 6× SDS-sample buffer. The mixture was heated for 5 min at 100°C. Fifteen microlitre samples were subjected to SDS-PAGE and immunoblotting. The VSV pseudotyped virus was prepared with the same procedure and used as the pseudovirus negative control, cell culture medium as negative control. The incorporation of the spike protein on the pseudovirus surface was confirmed using Western bolt with SARS-CoV-2 convalescent serum sample as the detection antibody with a 500-fold dilution. Goat anti-human IgG (Jackson ImmunoResearch, 109-035-0030) was used with a 1:8000 dilution as the secondary antibody.
anti-IgG
Buffers
Cell Culture Techniques
Cells
Centrifugation
Culture Media
Goat
Homo sapiens
Immunoglobulins
M protein, multiple myeloma
Pellets, Drug
SARS-CoV-2
SDS-PAGE
Serum
Sucrose
Technique, Dilution
Virus
Most recents protocols related to «Western blot»
Western blot procedures were performed as described in ref [36 (link)]. Antibodies employed for Western blot are detailed in Table S1 .
Western blot was performed according to our previous paper [4 (link)]. In brief, whole cell extracts were prepared by lysing the cells in lysis buffer (KeyGEN biotech, Nanjing, China). Then the equal amounts of total proteins were resolved by SDS-PAGE and the proteins of interest were probed by Western blot. Subsequently the Western blot results were visualized by enhanced chemiluminescence according to manufacturer’s instructions (Millipore, Billerica, MA, USA). The expression of protein was quantified by Image J software.
Total cellular proteins were extracted from cells or liver samples. The protein analysis by western blot was as described previously [20 (link)]. Protein bands were detected and analyzed using a ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, CA, USA). β-actin was used as a loading control. Results were expressed as the integrated optical density relative to β-actin or GAPDH. Full scans of western blot assays are shown in Additional file 1 : Figs. S1–S4.
Western blot analysis was performed as described previously [9] (link), BGN (1:500, 16409-1-AP, Proteintech, USA), N-cadherin (1:200, sc-59987, Santa Cruz, USA), Vimentin (1:200, sc-373717, Santa Cruz, USA), β-actin (1: 5000, Cell Signalling Technology) were utilized in western blot to check invasive capacity of glioma cells. SRY-Box Transcription Factor 2(SOX2, Cell Signalling Technology) and β-catenin (Cell Signalling Technology) was used to test expression stemness marker of TMZ treated cells.
The cell lysate was mixed with a non-reducing lane marker sample buffer, followed by heat denaturation. A Bradford assay was performed to quantify the protein concentration, and 20 µg of protein was extracted from each sample for western blot analysis. The protein was applied to a 10% SDS-polyacrylamide gel, transferred to polyvinylidene fluoride (PVDF) membranes (TermoFisher), and then detected by proper primary and secondary antibodies before visualization by the ECL western blot kit. The visualized bands were exposed using a gel imaging system, and the results were analysed using ImageJ software.
Top products related to «Western blot»
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PVDF membranes are a type of laboratory equipment used for a variety of applications. They are made from polyvinylidene fluoride (PVDF), a durable and chemically resistant material. PVDF membranes are known for their high mechanical strength, thermal stability, and resistance to a wide range of chemicals. They are commonly used in various filtration, separation, and analysis processes in scientific and research settings.
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RIPA lysis buffer is a detergent-based buffer solution designed for the extraction and solubilization of proteins from cells and tissues. It contains a mixture of ionic and non-ionic detergents that disrupt cell membranes and solubilize cellular proteins. The buffer also includes additional components that help to maintain the stability and activity of the extracted proteins.
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Protease inhibitor cocktail is a laboratory reagent used to inhibit the activity of proteases, which are enzymes that break down proteins. It is commonly used in protein extraction and purification procedures to prevent protein degradation.
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The BCA protein assay kit is a colorimetric-based method for the quantitative determination of total protein concentration in a sample. It uses bicinchoninic acid (BCA) to detect and quantify the presence of protein.
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The BCA Protein Assay Kit is a colorimetric detection and quantification method for total protein concentration. It utilizes bicinchoninic acid (BCA) for the colorimetric detection and quantification of total protein. The assay is based on the reduction of Cu2+ to Cu1+ by protein in an alkaline medium, with the chelation of BCA with the Cu1+ ion resulting in a purple-colored reaction product that exhibits a strong absorbance at 562 nm, which is proportional to the amount of protein present in the sample.
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β-actin is a protein that is found in all eukaryotic cells and is involved in the structure and function of the cytoskeleton. It is a key component of the actin filaments that make up the cytoskeleton and plays a critical role in cell motility, cell division, and other cellular processes.
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The Protease Inhibitor Cocktail is a laboratory product designed to inhibit the activity of proteases, which are enzymes that can degrade proteins. It is a combination of various chemical compounds that work to prevent the breakdown of proteins in biological samples, allowing for more accurate analysis and preservation of protein integrity.
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Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA and small interfering RNA (siRNA), into a wide range of eukaryotic cell types. It facilitates the formation of complexes between the nucleic acid and the lipid components, which can then be introduced into cells to enable gene expression or gene silencing studies.
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PVDF membranes are a type of laboratory equipment used for protein transfer and detection in Western blot analysis. They provide a stable and durable surface for the immobilization of proteins, enabling effective identification and quantification of target proteins in complex biological samples.
Sourced in China, United States, Switzerland, Germany, United Kingdom
RIPA buffer is a detergent-based cell lysis and extraction reagent. It is used to extract and solubilize proteins from cells and tissues for analysis. The buffer contains a combination of ionic and non-ionic detergents, as well as other components that aid in the solubilization and stabilization of proteins.
More about "Western blot"
Western blotting (immunoblotting) is a widely used analytical technique in molecular biology and biochemistry for the detection and quantification of specific proteins within a complex mixture extracted from cells or tissues.
This process involves several key steps: 1.
Protein separation: Proteins are first separated by size using gel electrophoresis, typically SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). 2.
Protein transfer: The separated proteins are then transferred from the gel to a membrane, often a PVDF (polyvinylidene difluoride) or nitrocellulose membrane, using electroblotting. 3.
Protein detection: The membrane is then incubated with specific primary antibodies that recognize the target protein of interest.
After washing, a secondary antibody conjugated with a reporter molecule (e.g., enzyme or fluorescent dye) is added to bind to the primary antibody.
This allows the visualization and quantification of the target protein.
The RIPA (radioimmunoprecipitation assay) lysis buffer is commonly used to extract proteins from cells or tissues, as it helps to solubilize and denature proteins.
Protease inhibitor cocktails are often added to the lysis buffer to prevent protein degradation.
The BCA (bicinchoninic acid) protein assay kit is a widely used method for determining the concentration of proteins in a sample.
Lipofectamine 2000 is a transfection reagent commonly used to introduce genetic material, such as DNA or RNA, into cells, which can be useful for studying the effects of protein overexpression or knockdown on Western blot results.
The housekeeping protein β-actin is frequently used as a loading control in Western blot analysis, as its expression is generally consistent across different samples and conditions.
PubCompare.ai is an innovative platform that leverages advanced machine learning to help researchers optimize their Western blot protocols.
By allowing users to easily locate and compare published methods, PubCompare.ai provides data-driven insights to help achieve consistent, high-quality results and take the guesswork out of the Western blot workflow.
This process involves several key steps: 1.
Protein separation: Proteins are first separated by size using gel electrophoresis, typically SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). 2.
Protein transfer: The separated proteins are then transferred from the gel to a membrane, often a PVDF (polyvinylidene difluoride) or nitrocellulose membrane, using electroblotting. 3.
Protein detection: The membrane is then incubated with specific primary antibodies that recognize the target protein of interest.
After washing, a secondary antibody conjugated with a reporter molecule (e.g., enzyme or fluorescent dye) is added to bind to the primary antibody.
This allows the visualization and quantification of the target protein.
The RIPA (radioimmunoprecipitation assay) lysis buffer is commonly used to extract proteins from cells or tissues, as it helps to solubilize and denature proteins.
Protease inhibitor cocktails are often added to the lysis buffer to prevent protein degradation.
The BCA (bicinchoninic acid) protein assay kit is a widely used method for determining the concentration of proteins in a sample.
Lipofectamine 2000 is a transfection reagent commonly used to introduce genetic material, such as DNA or RNA, into cells, which can be useful for studying the effects of protein overexpression or knockdown on Western blot results.
The housekeeping protein β-actin is frequently used as a loading control in Western blot analysis, as its expression is generally consistent across different samples and conditions.
PubCompare.ai is an innovative platform that leverages advanced machine learning to help researchers optimize their Western blot protocols.
By allowing users to easily locate and compare published methods, PubCompare.ai provides data-driven insights to help achieve consistent, high-quality results and take the guesswork out of the Western blot workflow.