This document discusses different electrophoresis techniques for analyzing protein-protein interactions, specifically blue-native electrophoresis (BNE), clear native electrophoresis (CNE), and high-resolution clear native electrophoresis (hrCNE). BNE uses Coomassie dye to impart a negative charge on proteins, allowing membrane proteins to migrate toward the anode. CNE uses milder conditions at neutral pH to isolate native membrane protein complexes. hrCNE uses anionic detergent micelles instead of dye. BNE provides the best resolution but CNE is milder and better preserves labile complexes. Two-dimensional native electrophoresis can further resolve complexes into individual components. Together these techniques allow isolation and characterization of both stable and transient
Gel Based Proteomics and Protein Sequences AnalysisGelica F
Two-dimensional gel electrophoresis (2DE) is the standard method for quantitative proteome analysis. It combines protein separation based on isoelectric focusing and molecular weight. In the first dimension, proteins are separated based on their isoelectric point using immobilized pH gradients. In the second dimension, proteins are separated by molecular weight using SDS-PAGE. The separated protein spots are then analyzed using mass spectrometry to identify individual proteins. 2DE provides high resolution and the ability to analyze thousands of proteins simultaneously, but it also has limitations including irreproducibility and inability to resolve all proteins.
This document discusses two-dimensional gel electrophoresis (2DGE), a powerful technique for analyzing complex protein mixtures. 2DGE separates proteins in two steps - first by isoelectric focusing to separate proteins by their isoelectric point, followed by SDS-PAGE to separate by molecular weight. This allows thousands of proteins to be separated simultaneously on a single gel based on these two properties. The document provides details on the principles, protocols, and improvements of the 2DGE technique.
The document describes the process of western blotting, which involves three main steps:
1. Proteins are extracted from cells and separated by gel electrophoresis based on their size.
2. Proteins are transferred from the gel to a membrane and detected using specific primary and secondary antibodies that bind to the target protein.
3. An enzyme-linked secondary antibody is used to visualize the target protein on the membrane through a detection method such as staining or imaging on an X-ray film. Western blotting is widely used to detect specific proteins in biological samples.
This document discusses various methods for analyzing proteins, including qualitative and quantitative analysis. Qualitative methods include Sanger's reaction, Edman's reaction, reaction with dansyl chloride, and others to identify amino acid composition. Quantitative methods discussed include Kjeldahl method, Dumas method, Biuret method, Lowry method, and spectroscopic analysis using UV/visible light absorption. Purification methods described are based on molecular size, solubility differences, and electric charges using techniques like electrophoresis, gel filtration, and ammonium sulfate precipitation.
Electrophoresis principle and types by Dr. Anurag YadavDr Anurag Yadav
the general principle on how the electrophoresis performs.
the different types of electrophoresis and the mechanism of separation based on different character of the medium and type of electrophoresis.
electrophoresis-
principle
types
details on paper electrophoresis
cellulose acetate electrophoresis
zone electrophoresis
SDS-PAGE
iso-electric focussing gel electrophoresis
two-dimensional gel electrophoresis
pulsed gel electrophoresis
isotachophoresis
capillary electrophoresis
microchip electrophoresis
Gel Based Proteomics and Protein Sequences AnalysisGelica F
Two-dimensional gel electrophoresis (2DE) is the standard method for quantitative proteome analysis. It combines protein separation based on isoelectric focusing and molecular weight. In the first dimension, proteins are separated based on their isoelectric point using immobilized pH gradients. In the second dimension, proteins are separated by molecular weight using SDS-PAGE. The separated protein spots are then analyzed using mass spectrometry to identify individual proteins. 2DE provides high resolution and the ability to analyze thousands of proteins simultaneously, but it also has limitations including irreproducibility and inability to resolve all proteins.
This document discusses two-dimensional gel electrophoresis (2DGE), a powerful technique for analyzing complex protein mixtures. 2DGE separates proteins in two steps - first by isoelectric focusing to separate proteins by their isoelectric point, followed by SDS-PAGE to separate by molecular weight. This allows thousands of proteins to be separated simultaneously on a single gel based on these two properties. The document provides details on the principles, protocols, and improvements of the 2DGE technique.
The document describes the process of western blotting, which involves three main steps:
1. Proteins are extracted from cells and separated by gel electrophoresis based on their size.
2. Proteins are transferred from the gel to a membrane and detected using specific primary and secondary antibodies that bind to the target protein.
3. An enzyme-linked secondary antibody is used to visualize the target protein on the membrane through a detection method such as staining or imaging on an X-ray film. Western blotting is widely used to detect specific proteins in biological samples.
This document discusses various methods for analyzing proteins, including qualitative and quantitative analysis. Qualitative methods include Sanger's reaction, Edman's reaction, reaction with dansyl chloride, and others to identify amino acid composition. Quantitative methods discussed include Kjeldahl method, Dumas method, Biuret method, Lowry method, and spectroscopic analysis using UV/visible light absorption. Purification methods described are based on molecular size, solubility differences, and electric charges using techniques like electrophoresis, gel filtration, and ammonium sulfate precipitation.
Electrophoresis principle and types by Dr. Anurag YadavDr Anurag Yadav
the general principle on how the electrophoresis performs.
the different types of electrophoresis and the mechanism of separation based on different character of the medium and type of electrophoresis.
electrophoresis-
principle
types
details on paper electrophoresis
cellulose acetate electrophoresis
zone electrophoresis
SDS-PAGE
iso-electric focussing gel electrophoresis
two-dimensional gel electrophoresis
pulsed gel electrophoresis
isotachophoresis
capillary electrophoresis
microchip electrophoresis
This document describes the process of preparing and isolating genomic DNA from bacterial cells. It involves 4 main steps:
1) Growing and harvesting bacterial cells in nutrient broth media. Common media used are M9 and Luria-Bertani broth.
2) Preparing a cell extract by lysing the bacterial cells using enzymes like lysozyme and detergents like SDS.
3) Purifying the DNA from other cell components like proteins and RNA. This is done using phenol-chloroform extraction and protease/RNase digestion. Ion-exchange chromatography can also be used.
4) Concentrating the purified DNA using ethanol precipitation, which causes the long DNA strands to precipitate out of
Chromatofocusing is a protein separation technique that uses ion exchange resins and buffers with changing pH to separate proteins based on their isoelectric point. As the buffer pH passes through a protein's pI, the protein loses its charge and elutes from the resin. Chromatofocusing provides high resolution separation of proteins that have similar pI values. However, some proteins may aggregate at high concentrations and clog the resin. Isoelectric focusing uses immobilized pH gradients in gels to separate proteins based on their pI through electrophoresis. Two-dimensional electrophoresis separates proteins first by pI using isoelectric focusing, then by molecular weight using SDS-PAGE to provide high resolution separation and identification of
METHODS TO DETERMINE PROTEIN STRUCTURE Sabahat Ali
This document discusses several methods for determining protein structure: Edman degradation, X-ray crystallography, Western blotting, SDS-PAGE, 2D gel electrophoresis, and isoelectric focusing. Edman degradation involves chemically removing amino acids from the N-terminus of a protein one by one to determine the sequence. X-ray crystallography provides high-resolution 3D structures of proteins. Western blotting identifies specific proteins in a sample using antibodies. SDS-PAGE and 2D gels separate proteins by size and electric charge properties. Isoelectric focusing separates proteins based on their isoelectric points.
This document describes methods for purifying DNA from living cells. It discusses purification of total cellular DNA, plasmid DNA, and bacteriophage DNA. The basic protocol involves lysing cells to release DNA, then using enzymatic or chemical treatments to remove contaminating proteins, RNA, and other molecules. Specific techniques described include phenol/chloroform extraction, ion exchange chromatography, alkaline denaturation, and CsCl gradient centrifugation. The goal is to obtain purified DNA samples suitable for downstream applications like PCR and sequencing.
Western Blotting (Protein Separation technique) .pptxAnkit Mehra
The Western blot (Immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecting the proteins, this technique is also utilized to visualize, distinguish and quantify the different proteins in a complicated protein combination.
This slide share is meant to explain the principle, process and some minute details but the best way to understand any analytical technique is by performing it labs and doing several troubleshooting.
This document provides an overview of electrophoresis, including:
1. Electrophoresis uses the migration of charged solutes or particles in a liquid medium under the influence of an electric field. It is widely used to separate biological molecules like proteins.
2. Particles with different charge-to-mass ratios migrate at different rates depending on factors like their net charge, size, and the pH and strength of the buffer solution. Agarose gel and polyacrylamide gel electrophoresis are commonly used techniques.
3. The general procedure involves separating the particles in an electric field, staining to visualize the bands, then detecting and quantifying the separated fractions. Automated systems now allow high-throughput processing of
Western blotting is a technique used to detect specific proteins in a sample:
1) Proteins are first separated by electrophoresis and then transferred to a membrane for detection.
2) Antibodies are used to detect the target protein(s) on the membrane through binding.
3) An enzyme-linked secondary antibody is used to visualize the bound primary antibodies, allowing visualization of bands that correspond to the target proteins.
This document provides information on electrophoresis, including:
1. Electrophoresis is a technique used to separate charged particles like proteins and nucleic acids in an electric field based on their charge-to-mass ratio.
2. Agarose and polyacrylamide gels are commonly used supporting media that provide a matrix for particle separation.
3. The general procedure involves sample application, electrophoretic separation, staining to visualize bands, and detection/quantification of bands through densitometry.
2D gel electrophoresis separates proteins based on their isoelectric point (pI) in the first dimension and molecular weight in the second dimension. Amino acids can exist as zwitterions and act as both acids and bases, donating or accepting protons. The isoelectric point is calculated using the Henderson-Hasselbalch equation and titration curves, taking into account the pKa values of amino acid side chains. Detection methods for proteins on 2D gels include staining with dyes or radioactive labeling.
The document describes electrosomes, which are a novel surface display system composed of enzymes attached to a scaffoldin protein. This allows for multiple electron release from fuel oxidation. In the anode, an ethanol oxidation cascade is assembled using alcohol dehydrogenase and formaldehyde dehydrogenase enzymes attached to the scaffoldin. In the cathode, copper oxidase is attached for oxygen reduction. The electrosomes provide advantages as a fuel cell and drug delivery system by catalyzing chemical energy conversion to electricity and providing controlled drug release.
The document discusses methods for preparing tissue or cell extracts for protein separation and analysis. It describes various cell lysis buffers and their uses depending on the protein location. It also discusses steps to inhibit protein degradation during extraction, such as using protease inhibitors and reducing agents. The document compares the Lowry and Bradford methods for estimating protein concentration, noting the principles, advantages, and disadvantages of each. It also discusses the importance of trichloroacetic acid precipitation to separate proteins from interfering substances.
Buffers resist changes in pH and consist of a conjugate acid-base pair where the ratio of proton acceptor to proton donor is near unity. A webinar discusses how biological buffers can impact reproducibility and explores considerations for buffer selection and use to improve reproducibility. Key factors include how buffers may interact with metals and biological materials and tips for proper buffer selection and use. [END SUMMARY]
Exploring Proteins and Proteomes. Stryer,CHAPTER 3 pptkhair ullah
Methods in Protein Chemistry
This chapter discusses several methods used to isolate, purify, detect, degrade, analyze, and synthesize proteins. It describes techniques such as centrifugation, solubility, dialysis, gel filtration, affinity chromatography, HPLC, electrophoresis, and mass spectrometry. It also covers determining a protein's amino acid sequence through methods like Edman degradation, solid phase synthesis, chemical and enzymatic cleavage, and the use of DNA sequencing. The goal of these methods is to obtain a protein's amino acid sequence and gain functional information about proteins and proteomes.
Gel electrophoresis is a technique used to separate biomolecules like proteins and nucleic acids based on their size and charge. When placed in an electric field, charged biomolecules will migrate through a gel towards the electrode of opposite charge. Smaller molecules travel farther than larger ones. Common types of gels used are agarose for separating larger nucleic acids and polyacrylamide for separating smaller nucleic acids and proteins. Samples are loaded into wells and run in a buffer solution through the gel. After electrophoresis, the gel can be stained to visualize the separated biomolecules bands for analysis. Gel electrophoresis has various applications in fields like forensics, molecular biology, and biochemistry.
preparation of genomic DNA from bacteria.pdfAnjir Rumey
This document describes the process of preparing genomic DNA from bacteria. It involves four main steps: 1) growing and harvesting bacterial cells, 2) breaking open the cells to release DNA and other components, 3) purifying the DNA from other cell components through organic extraction and chromatography, and 4) concentrating the purified DNA. Key techniques include using lysozyme and EDTA to break open cell walls, phenol-chloroform extraction to remove proteins, and ethanol precipitation to concentrate DNA.
This document outlines a method for isolating genomic DNA from plant shoot tissue. The method involves grinding plant tissue, suspending it in extraction buffer, incubating, extracting with chloroform, precipitating the DNA with isopropyl alcohol, and re-suspending the DNA in buffer. On the second day, the isolated plant DNA is compared by gel electrophoresis to DNA isolated from chloroplasts using restriction enzyme digestion. The method allows isolation of nuclear, chloroplast, and mitochondrial DNA from plant cells. CTAB in the extraction buffer helps lyse cells and denature proteins while preserving DNA integrity for downstream applications.
Drug and gene delivery vehicles are biocompatible devices that can carry therapeutic components in the body. Synthetic vehicles include block copolymers, liposomes, dendrimers, and magnetic nanoparticles. Block copolymers form micelles with hydrophobic cores that can encapsulate drugs. Liposomes are phospholipid vesicles that can encapsulate both hydrophilic and hydrophobic drugs. Dendrimers are nanoscale polymers that can be functionalized to target drugs. Magnetic nanoparticles can be used for drug delivery, hyperthermia cancer treatment, and as MRI contrast agents. These vehicles aim to improve drug bioavailability and targeting while decreasing toxicity.
This document provides information on electrophoresis techniques. It discusses how electrophoresis separates charged molecules like proteins and nucleic acids using an electric current. The key techniques covered are:
1. SDS-PAGE, which uses sodium dodecyl sulfate to denature proteins and give them a uniform negative charge for separation by size in a polyacrylamide gel.
2. Native PAGE, which separates intact proteins by their charge-to-size ratio.
3. Isoelectric focusing, which separates proteins based on their isoelectric point in a pH gradient gel.
It also discusses two-dimensional electrophoresis, which combines isoelectric focusing and SDS-PAGE to better resolve complex protein mixtures. The document
Western blotting is a technique used to detect specific proteins in a sample. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a target protein based on its molecular weight and signal intensity. Key steps include sample preparation, electrophoresis to separate proteins by size, electrotransfer to a membrane, blocking to reduce background noise, probing with primary and secondary antibodies, washing, and detection of the target protein. The technique allows identification and quantification of proteins but has limitations related to its qualitative nature and specificity of antibodies used.
This study used next generation sequencing to identify and characterize a novel betanucleorhabdovirus infecting Cnidium officinale plants in South Korea. High throughput sequencing of RNA from infected plants yielded a 14kb viral genome, tentatively named Cnidium virus 1 (CnV1). The complete genome sequence of CnV1 was determined and found to have six open reading frames in the order 3'-N-P-P3-M-G-L-5', resembling other plant rhabdoviruses. This is the first report of CnV1 infecting C. officinale in Korea. Next generation sequencing was an efficient method for detecting this novel viral population.
This document summarizes a study that identified viral diseases affecting the Cnidium officinale plant in South Korea in fall 2021. Samples showing virus-like symptoms were collected and total RNA was extracted. RT-PCR was performed using primers for known viruses that infect cnidium. All samples tested positive for Apple stem grooving virus, Cnidium Vein Yellow viruses 1 and 2, and Cnidium virus X, indicating mixed infections with one or more of these four viruses. The complete genomic sequence of a new betanucleorhabdovirus found in the plants, tentatively named Cnidium virus 1, is also described.
More Related Content
Similar to Native electrophoretic techniques ppt.pptx
This document describes the process of preparing and isolating genomic DNA from bacterial cells. It involves 4 main steps:
1) Growing and harvesting bacterial cells in nutrient broth media. Common media used are M9 and Luria-Bertani broth.
2) Preparing a cell extract by lysing the bacterial cells using enzymes like lysozyme and detergents like SDS.
3) Purifying the DNA from other cell components like proteins and RNA. This is done using phenol-chloroform extraction and protease/RNase digestion. Ion-exchange chromatography can also be used.
4) Concentrating the purified DNA using ethanol precipitation, which causes the long DNA strands to precipitate out of
Chromatofocusing is a protein separation technique that uses ion exchange resins and buffers with changing pH to separate proteins based on their isoelectric point. As the buffer pH passes through a protein's pI, the protein loses its charge and elutes from the resin. Chromatofocusing provides high resolution separation of proteins that have similar pI values. However, some proteins may aggregate at high concentrations and clog the resin. Isoelectric focusing uses immobilized pH gradients in gels to separate proteins based on their pI through electrophoresis. Two-dimensional electrophoresis separates proteins first by pI using isoelectric focusing, then by molecular weight using SDS-PAGE to provide high resolution separation and identification of
METHODS TO DETERMINE PROTEIN STRUCTURE Sabahat Ali
This document discusses several methods for determining protein structure: Edman degradation, X-ray crystallography, Western blotting, SDS-PAGE, 2D gel electrophoresis, and isoelectric focusing. Edman degradation involves chemically removing amino acids from the N-terminus of a protein one by one to determine the sequence. X-ray crystallography provides high-resolution 3D structures of proteins. Western blotting identifies specific proteins in a sample using antibodies. SDS-PAGE and 2D gels separate proteins by size and electric charge properties. Isoelectric focusing separates proteins based on their isoelectric points.
This document describes methods for purifying DNA from living cells. It discusses purification of total cellular DNA, plasmid DNA, and bacteriophage DNA. The basic protocol involves lysing cells to release DNA, then using enzymatic or chemical treatments to remove contaminating proteins, RNA, and other molecules. Specific techniques described include phenol/chloroform extraction, ion exchange chromatography, alkaline denaturation, and CsCl gradient centrifugation. The goal is to obtain purified DNA samples suitable for downstream applications like PCR and sequencing.
Western Blotting (Protein Separation technique) .pptxAnkit Mehra
The Western blot (Immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecting the proteins, this technique is also utilized to visualize, distinguish and quantify the different proteins in a complicated protein combination.
This slide share is meant to explain the principle, process and some minute details but the best way to understand any analytical technique is by performing it labs and doing several troubleshooting.
This document provides an overview of electrophoresis, including:
1. Electrophoresis uses the migration of charged solutes or particles in a liquid medium under the influence of an electric field. It is widely used to separate biological molecules like proteins.
2. Particles with different charge-to-mass ratios migrate at different rates depending on factors like their net charge, size, and the pH and strength of the buffer solution. Agarose gel and polyacrylamide gel electrophoresis are commonly used techniques.
3. The general procedure involves separating the particles in an electric field, staining to visualize the bands, then detecting and quantifying the separated fractions. Automated systems now allow high-throughput processing of
Western blotting is a technique used to detect specific proteins in a sample:
1) Proteins are first separated by electrophoresis and then transferred to a membrane for detection.
2) Antibodies are used to detect the target protein(s) on the membrane through binding.
3) An enzyme-linked secondary antibody is used to visualize the bound primary antibodies, allowing visualization of bands that correspond to the target proteins.
This document provides information on electrophoresis, including:
1. Electrophoresis is a technique used to separate charged particles like proteins and nucleic acids in an electric field based on their charge-to-mass ratio.
2. Agarose and polyacrylamide gels are commonly used supporting media that provide a matrix for particle separation.
3. The general procedure involves sample application, electrophoretic separation, staining to visualize bands, and detection/quantification of bands through densitometry.
2D gel electrophoresis separates proteins based on their isoelectric point (pI) in the first dimension and molecular weight in the second dimension. Amino acids can exist as zwitterions and act as both acids and bases, donating or accepting protons. The isoelectric point is calculated using the Henderson-Hasselbalch equation and titration curves, taking into account the pKa values of amino acid side chains. Detection methods for proteins on 2D gels include staining with dyes or radioactive labeling.
The document describes electrosomes, which are a novel surface display system composed of enzymes attached to a scaffoldin protein. This allows for multiple electron release from fuel oxidation. In the anode, an ethanol oxidation cascade is assembled using alcohol dehydrogenase and formaldehyde dehydrogenase enzymes attached to the scaffoldin. In the cathode, copper oxidase is attached for oxygen reduction. The electrosomes provide advantages as a fuel cell and drug delivery system by catalyzing chemical energy conversion to electricity and providing controlled drug release.
The document discusses methods for preparing tissue or cell extracts for protein separation and analysis. It describes various cell lysis buffers and their uses depending on the protein location. It also discusses steps to inhibit protein degradation during extraction, such as using protease inhibitors and reducing agents. The document compares the Lowry and Bradford methods for estimating protein concentration, noting the principles, advantages, and disadvantages of each. It also discusses the importance of trichloroacetic acid precipitation to separate proteins from interfering substances.
Buffers resist changes in pH and consist of a conjugate acid-base pair where the ratio of proton acceptor to proton donor is near unity. A webinar discusses how biological buffers can impact reproducibility and explores considerations for buffer selection and use to improve reproducibility. Key factors include how buffers may interact with metals and biological materials and tips for proper buffer selection and use. [END SUMMARY]
Exploring Proteins and Proteomes. Stryer,CHAPTER 3 pptkhair ullah
Methods in Protein Chemistry
This chapter discusses several methods used to isolate, purify, detect, degrade, analyze, and synthesize proteins. It describes techniques such as centrifugation, solubility, dialysis, gel filtration, affinity chromatography, HPLC, electrophoresis, and mass spectrometry. It also covers determining a protein's amino acid sequence through methods like Edman degradation, solid phase synthesis, chemical and enzymatic cleavage, and the use of DNA sequencing. The goal of these methods is to obtain a protein's amino acid sequence and gain functional information about proteins and proteomes.
Gel electrophoresis is a technique used to separate biomolecules like proteins and nucleic acids based on their size and charge. When placed in an electric field, charged biomolecules will migrate through a gel towards the electrode of opposite charge. Smaller molecules travel farther than larger ones. Common types of gels used are agarose for separating larger nucleic acids and polyacrylamide for separating smaller nucleic acids and proteins. Samples are loaded into wells and run in a buffer solution through the gel. After electrophoresis, the gel can be stained to visualize the separated biomolecules bands for analysis. Gel electrophoresis has various applications in fields like forensics, molecular biology, and biochemistry.
preparation of genomic DNA from bacteria.pdfAnjir Rumey
This document describes the process of preparing genomic DNA from bacteria. It involves four main steps: 1) growing and harvesting bacterial cells, 2) breaking open the cells to release DNA and other components, 3) purifying the DNA from other cell components through organic extraction and chromatography, and 4) concentrating the purified DNA. Key techniques include using lysozyme and EDTA to break open cell walls, phenol-chloroform extraction to remove proteins, and ethanol precipitation to concentrate DNA.
This document outlines a method for isolating genomic DNA from plant shoot tissue. The method involves grinding plant tissue, suspending it in extraction buffer, incubating, extracting with chloroform, precipitating the DNA with isopropyl alcohol, and re-suspending the DNA in buffer. On the second day, the isolated plant DNA is compared by gel electrophoresis to DNA isolated from chloroplasts using restriction enzyme digestion. The method allows isolation of nuclear, chloroplast, and mitochondrial DNA from plant cells. CTAB in the extraction buffer helps lyse cells and denature proteins while preserving DNA integrity for downstream applications.
Drug and gene delivery vehicles are biocompatible devices that can carry therapeutic components in the body. Synthetic vehicles include block copolymers, liposomes, dendrimers, and magnetic nanoparticles. Block copolymers form micelles with hydrophobic cores that can encapsulate drugs. Liposomes are phospholipid vesicles that can encapsulate both hydrophilic and hydrophobic drugs. Dendrimers are nanoscale polymers that can be functionalized to target drugs. Magnetic nanoparticles can be used for drug delivery, hyperthermia cancer treatment, and as MRI contrast agents. These vehicles aim to improve drug bioavailability and targeting while decreasing toxicity.
This document provides information on electrophoresis techniques. It discusses how electrophoresis separates charged molecules like proteins and nucleic acids using an electric current. The key techniques covered are:
1. SDS-PAGE, which uses sodium dodecyl sulfate to denature proteins and give them a uniform negative charge for separation by size in a polyacrylamide gel.
2. Native PAGE, which separates intact proteins by their charge-to-size ratio.
3. Isoelectric focusing, which separates proteins based on their isoelectric point in a pH gradient gel.
It also discusses two-dimensional electrophoresis, which combines isoelectric focusing and SDS-PAGE to better resolve complex protein mixtures. The document
Western blotting is a technique used to detect specific proteins in a sample. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a target protein based on its molecular weight and signal intensity. Key steps include sample preparation, electrophoresis to separate proteins by size, electrotransfer to a membrane, blocking to reduce background noise, probing with primary and secondary antibodies, washing, and detection of the target protein. The technique allows identification and quantification of proteins but has limitations related to its qualitative nature and specificity of antibodies used.
Similar to Native electrophoretic techniques ppt.pptx (20)
This study used next generation sequencing to identify and characterize a novel betanucleorhabdovirus infecting Cnidium officinale plants in South Korea. High throughput sequencing of RNA from infected plants yielded a 14kb viral genome, tentatively named Cnidium virus 1 (CnV1). The complete genome sequence of CnV1 was determined and found to have six open reading frames in the order 3'-N-P-P3-M-G-L-5', resembling other plant rhabdoviruses. This is the first report of CnV1 infecting C. officinale in Korea. Next generation sequencing was an efficient method for detecting this novel viral population.
This document summarizes a study that identified viral diseases affecting the Cnidium officinale plant in South Korea in fall 2021. Samples showing virus-like symptoms were collected and total RNA was extracted. RT-PCR was performed using primers for known viruses that infect cnidium. All samples tested positive for Apple stem grooving virus, Cnidium Vein Yellow viruses 1 and 2, and Cnidium virus X, indicating mixed infections with one or more of these four viruses. The complete genomic sequence of a new betanucleorhabdovirus found in the plants, tentatively named Cnidium virus 1, is also described.
The document summarizes a study that identified viral diseases affecting tobacco plants in South Korea. Researchers collected tobacco plant samples showing viral symptoms from farms. They tested a pooled sample using RT-PCR and identified that it was infected with four viruses: tobacco ring spot virus, tobacco streak virus, tobacco necrosis virus, and tobacco mosaic virus. The viruses identified infect a wide range of host plants and can cause significant damage and yield loss to crops like tobacco and tomatoes.
The student conducted field research to identify viral diseases affecting tobacco plants in South Korea. RT-PCR analysis of pooled tobacco plant samples showed infection by four viruses: tobacco ringspot virus, tobacco streak virus, tobacco necrosis virus, and tobacco mosaic virus. The student collected samples exhibiting viral symptoms from tobacco fields and tested them using RT-PCR to detect seven common tobacco viruses. The results identified that the pooled sample was infected with tobacco ringspot virus, tobacco streak virus, tobacco necrosis virus, and tobacco mosaic virus.
This report details a field research study identifying viral diseases affecting barley crops in South Korea. RT-PCR tests showed that samples were infected with both Barley yellow dwarf virus-PAS and Hordeum vulgare alphaendornavirus. The high occurrence of viruses during the spring season was likely due to suitable climate conditions for the distribution of viral vectors like aphids. A new endornavirus, named Hordeum vulgare alphaendornavirus Buan, was also identified that was 98% identical to a previously reported endornavirus in Korea. The study helps better understand the impact and prevalence of these viruses in barley crops in the Buan region.
This document discusses serological approaches for detecting COVID-19 through immune-based detection of antibodies. It provides an overview of the SARS-CoV-2 virus, current diagnostic methods including RT-PCR and immunoassays, considerations around specificity and sensitivity of different viral targets for immunoassays, applications of immunoassays for surveillance and developing treatments, and the need for point-of-care tests. Moving forward, it emphasizes that collating immunoassay data and developing rapid decentralized tests can help address testing shortages, while ensuring rigorous evaluation of new tests.
This document summarizes a paper on plant-microbe interactions under climate change. It discusses how climate change impacts the assembly of the plant microbiome through direct effects on microbes and indirect effects via changes to plants. Rising temperatures and drought alter pathogen abundance and benefical plant-microbe interactions. The paper also examines the evolutionary and eco-evolutionary responses of plant-microbiome interactions to climate change over short and long time scales. It concludes that manipulating the plant microbiome through breeding and genetic tools shows promise to increase plant resilience but more research is still needed to understand indirect impacts and long-term responses under climate change.
This document summarizes the discovery and analysis of two novel poleroviruses found infecting tobacco plants showing symptoms of tobacco bushy top disease (TBTD) in China. Next-generation sequencing was used to identify viral sequences present in two infected tobacco samples. Bioinformatics analysis revealed the presence of six viruses, including Tobacco bushy top virus, Tobacco vein distorting virus, their satellite RNAs, and two novel poleroviruses termed Tobacco polerovirus 1 and 2. Further analysis of the genomic sequences and phylogenetic trees showed that the two novel viruses are distinct from other known poleroviruses. RT-PCR detection confirmed the viruses in the original samples and additional field samples. This study expands knowledge
This document summarizes the development and validation of a one-step reverse transcription digital PCR (RT-dPCR) assay for the detection of SARS-CoV-2. Key points:
1. RT-dPCR was found to significantly improve the sensitivity of detection of SARS-CoV-2 in pharyngeal swab samples compared to RT-qPCR, reducing the false negative rate. RT-dPCR detected SARS-CoV-2 in 61 samples that were negative or equivocal by RT-qPCR.
2. When tested on 196 clinical samples, RT-dPCR increased the positive detection rate to 91% compared to 28% for RT-qPCR. RT-dPCR is well-su
1. The document discusses separation science techniques used in the author's laboratory to detect plant viruses, including liquid nitrogen grinding of plant samples, centrifugation-based nucleic acid extraction, PCR amplification, gel electrophoresis, and next generation sequencing.
2. Key separation techniques used are centrifugation to extract nucleic acids from plant samples, PCR to amplify viral DNA and RNA, gel electrophoresis to separate DNA fragments by size, and next generation sequencing to identify known and unknown viruses.
3. The overall work flow involves collecting plant samples, extracting and sequencing nucleic acids, assembling contigs, identifying virus sequences through database alignment and phylogenetic analysis to detect and identify viral populations in plants.
1. The plant microbiome varies significantly depending on the plant compartment, with rhizosphere, endophytes, and phyllosphere harboring distinct bacterial and fungal communities. Core and hub microbiota that are consistently present play important regulatory roles.
2. Plant colonization is initiated through chemotaxis towards root exudates, with microbes attaching to form biofilms. Community assembly involves dispersal, species interactions, environmental factors and host genetics.
3. The plant microbiome confers key functions like nutrient acquisition, disease resistance, and stress tolerance through mechanisms like nitrogen fixation, antimicrobial production, hormone modulation and phenology alteration. However, knowledge gaps around other microorganism types and breeding
This document summarizes the complete genomic sequence of a novel betanucleorhabdovirus identified from a Cnidium officinale plant and tentatively named Cnidium virus 1 (CnV1). The genome of CnV1 was sequenced and found to be 13,994 nucleotides in length. BLAST searches showed CnV1 is most closely related to betanucleorhabdoviruses. Several viruses have previously been reported to infect C. officinale including two secoviruses and an alphaflexivirus. This represents the first report of a betanucleorhabdovirus infecting C. officinale.
The document discusses centrifugation, which uses centrifugal force to separate mixtures based on density. It separates fluids and particles by spinning them at high speeds in a centrifuge. Centrifugation is commonly used in biology to isolate cellular components like organelles, DNA, RNA, and proteins. It separates mixtures into a pellet of dense particles and a supernatant of less dense fluid. Centrifugation has many applications and works by accelerating denser substances outward using sedimentation. Safety is important when operating centrifuges.
Real-time PCR, also known as quantitative real-time PCR, allows for both the amplification and simultaneous quantification of a targeted DNA molecule. It works by detecting the accumulation of a fluorescent signal during each cycle of the PCR reaction. Specifically, it relies on all the components of standard PCR as well as a fluorescent oligonucleotide probe containing reporter and quencher dyes - when the probe binds to the target DNA during amplification, the reporter dye produces a detectable fluorescent signal. This allows for the reaction to be continuously monitored in real time, providing both detection and quantification of the DNA target.
The document discusses various techniques and applications of polymerase chain reaction (PCR). It describes the invention of PCR in 1982 and some key adaptations such as real-time PCR, which allows for quantitative analysis of DNA amplification in real time using fluorescent probes. Different types of PCR are also summarized, including reverse transcription PCR, nested PCR, multiplex PCR, and touchdown PCR. Components of real-time PCR and steps in the PCR process are outlined.
This document summarizes a study on the effects of chitosan and chlorocholine chloride on cocoyam minituberization. The study found that applying chitosan at concentrations of 1, 2, and 3 g/L and chlorocholine chloride at 5, 10, and 15 mg/L significantly increased average plant height, number of leaves, and leaf surface area compared to the control. A two-way ANOVA and Tukey's multiple comparisons test showed highly significant interactions between the factors and differences between the means of most treatment combinations. The column factor of chitosan and chlorocholine chloride treatment had the strongest effect on the results.
This document summarizes seven strategies that plant viruses use to translate multiple proteins from a single mRNA, which is necessary because the host cell's translation system normally produces only one protein per mRNA. The strategies described are: 1) having a multipartite genome with multiple RNA segments, 2) producing proteins from a polyprotein via proteolytic cleavage, 3) using subgenomic RNAs, 4) read-through of stop codons to produce extended proteins, 5) leaky scanning of ribosomes, 6) internal initiation of translation, and 7) frameshifting during translation. Examples are provided for many plant viruses that employ each strategy, such as bromoviruses having multipartite genomes and potyviruses expressing via a poly
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Nucleophilic Addition of carbonyl compounds.pptxSSR02
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Travis Hills' Endeavors in Minnesota: Fostering Environmental and Economic Pr...Travis Hills MN
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Or: Beyond linear.
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Disclaimer: No one is perfect, so please mind that there might be mistakes and typos.
dtubbenhauer@gmail.com
Corrected slides: dtubbenhauer.com/talks.html
hematic appreciation test is a psychological assessment tool used to measure an individual's appreciation and understanding of specific themes or topics. This test helps to evaluate an individual's ability to connect different ideas and concepts within a given theme, as well as their overall comprehension and interpretation skills. The results of the test can provide valuable insights into an individual's cognitive abilities, creativity, and critical thinking skills
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1. University of Science and Technology,
Korean Research Institute of Bioscience and Biotechnology
Course Instructor: Ji-Seon Jeong
Presenter: Mesele
2. OUTLINE
Blue-Native Electrophoresis
1. Introduction
2. Identification of protein–protein interactions
Clear Native Electrophoresis
high-resolution Clear-Native Electrophoresis
Isolation of stable protein complexes by native electrophoresis
3. Introduction
Permanent protein–protein interactions are commonly
identified by co-purification of two /more protein components using techniques like
-co-immunoprecipitation,
-tandem affinity purification and
-native electrophoresis.
Hints for dynamic protein–protein interactions can be obtained using two-hybrid
techniques but not from native electrophoresis
4. Native
Electrophoresis
Blue-Native
Electrophoresis (BNE)
Clear Native
Electrophoresis (CNE)
high-resolution Clear-Native
Electrophoresis (hrCNE)
variants of the same basic technique that
differ by the cathode buffers used
Even minor variation of the cathode buffer
changes the separation principles and
affects resolution
The protein at the pH of the going for
buffer
Used to isolate
hydrophobic and water-soluble protein
larger membrane protein complexes and
supercomplexes, Fig.1.
identify stable membrane protein complexes
and some detergent-labile physiological super
complexes.
5. Starts with solubilization of biological membranes by neutral detergents and
insoluble material removed by centrifugation.
The supernatant directly applied to the gel and run at pH 7.
Proteins migrate according to their intrinsic pI,
only acidic proteins migrate to anode.
All proteins with pI>7 migrate to the cathode and are lost.
Smearing observed with many membrane proteins.
Offers special advantages for in-gel catalytic activity assays & fluorescent-labeled protein detection.
CNE is milder than BNE and
currently the mildest electrophoretic technique to isolate supramolecular structures
Clear native electrophoresis (CNE)
6. A single step isolation of membrane protein complexes from purified mitochondria.
Use mild neutral detergents for membrane solubilization like CNE
bis-tris/imidazole buffers: applied to stabilize pH to 7.0–7.5 in the native gels.
Coomassie Brillant Blue G-250 (anionic dye): to impose (-) charge shift on proteins upon binding to protein surface.
The charge shift is important for hydrophobic membrane proteins, reduces protein aggregation
All Coomassie-binding membrane proteins migrate to anode independent of the protein intrinsic pI.
Many water soluble proteins, pI>7 do not bind the dye. cathodic migration, lost with the cathode buffer.
A disadvantageous aspect of the dye
in the presence of neutral detergents, initial protein solubilization: detergent micelles are formed dissociate
detergent-labile subunits from multiprotein complexes disassemble supramolecular structures.
Blue-native electrophoresis (BNE)
7. -ve charge
Hydrophobic chain
Dimethyl base –enables to
bind a protein
Native protein electrophoresis: separates acidic water-soluble and membrane proteins in a polyacrylamide
gradient gel. It uses no charged dye so the electrophoretic mobility of proteins in CN-PAGE (in contrast to the
charge shift technique BN-PAGE) is related to the intrinsic charge of the proteins.
8. Developed to preserve the advantages of BNE & CNE and eliminate their disadvantages
Closer to BNE, since hrCNE is a charge shift technique and resolution is comparable to BNE.
Instead of Coomassie-dye,
non-colored mixed micelles of neutral and
anionic detergents are used to induce the charge shift on proteins.
Have three variants, hrCNE1–3, differing in the composition of mixed anionic detergent micelles.
Promote dissociation of labile subunits from protein complexes and
considered less mild than CNE and even harsher than BNE.
Overall, BNE is the most robust variant that is preferable to hrCNE and CNE except for analyses of
fluorescent-labeled proteins or in-gel catalytic activity assays.
CNE, the mildest variant, is specifically useful for the analysis of physiological supramolecular structures
provided the limitations in resolution are acceptable.
High-resolution clear-native electrophoresis (hrCNE)
9.
10. 3–13% acrylamide gradient gels were used as indicated. I-IV, respiratory complexes I–IV that used as native mass standards (masses indicated the right side).
M, D, T, H= monomeric, dimeric, tetrameric, hexameric forms of ATP synthase (complex V); F1= catalytic part of ATP synthase.
Using digitonin for solubilization of mitochondria (4 g/g protein), two supercomplexes (S0 and S1) of the respiratory chain containing complex I, complex III,
and 0 or 1 copy of complex IV were observed. They were optimally preserved in CN gels (lane Di) however, gave rise to extreme smearing of bands.
Supercomplexes S0 and S1 and the oligomeric forms of ATP synthase (D, T, H) were also preserved in BN and hrCNE-3 gels & occasionally hrCNE-1.
Setting dodecylmaltoside/protein and Triton X-100/protein ratios to 2.4 and 3 g/g, respectively, no supercomplexes but the individual complexes were
solubilized from mitochondrial membranes.
BNE is the most robust and versatile technique & is mostly used except for in-gel activity assays and detection of fluorescent proteins in 1-D native gels.
USING bovine heart
mitochondria solubilized by
digitonin (Di),
dodecylmaltoside (Do) and
Triton-X-100 (TX).
Comparative presentation of the native electrophoresis variants
11. 1-D BN and 2-D BN/SDS gels and blots, and recovery of proteins from BN gels
BN-PAGE)isolate membrane protein complexes immediately from biological membranes.
Native protein extracts: used for catalytic activity measurements & currently for advanced structural investigations.
Electroblotting of 1-D BN and 2-D BN/SDS gels and immunological detection of proteins by specific antibodies
became widely used techniques, especially when low abundant proteins are to be analyzed or the available protein
amounts are limiting,
e.g. when analyzing patient biopsy specimens or cultured human cells.
Use of 2-D BN/SDS-PAGE for native mass estimation of membrane
protein complexes.
(A) 2-D BN/SDS-PAGE of bovine mitochondrial complexes solubilized by
digitonin and separated by BNE
(B) Bovine respiratory complexes I–IV were used as mass standards (black
squares, masses indicated in right side) and plotted vs their migration
distances in BNE (arrow on top of Fig.A).
Estimated masses for
monomeric complex V (M)= 700 kDa,
dimeric complex V (D)= 1500 kDa, and
Super complexes S0 = 1500 kDa and S1= 1700 kDa
12. Identification of protein–protein interactions
A Coomassie-stained hrCNE-1 gel.
In-gel activity assays for respiratory
B complex I,
C complex II,
D complex III and
E complex IV.
F In-gel ATPase assay of complex V in the absence oligomycin or
G in the presence of oligomycin, an inhibitor of fully assembled ATP synthase.
H2-D BN/BN gel of solubilized bovine mitochondria using 1-D BNE for
separation of supercomplexes and
2-D modified BNE (with 0.02% dodecylmaltoside added to the cathode buffer)
for the release of individual complexes.
Respiratory supercomplexes S0 and S1 released the constituent complexes I, III
and IV.
Dimeric complex V (D)- dissociated into the monomeric complexes (M), and
released also small amounts of the catalytic F1 domain.
I Spots of individual complexes, e.g. complex V (boxed red), further analyzed by
dSDS-PAGE, i.e. by 3-D and 4-D SDS gels.
Complex V from yeast, cut out from a 2-D BN/BN gel similar to figure part H,
was first resolved by 3-D tricine-SDS-PAGE.
All subunits of complex V, except subunits c and a, migrated as individual bands
in the 3-D SDS gel, since only one spot each for the indicated individual subunits
was revealed after the 4-D separation.
Multidimensional gels to identify and characterize protein–protein interactions. (A–G) Detection of bovine mitochondrial complexes in 1-D hrCNE-1 gels.
13. The isolation requirements are very similar to the requirements for chromatographic preparations.
The most important factors are - Ionic strength, pH, Choice of detergent for membrane protein complexes
1. Ionic strength
low ionic strength: - support membrane protein solubilization by detergents and
- to keep some water-soluble proteins in solution.
‘‘Salting in’’ effects favoring protein solubilization observed at low ionic strength, e.g. 50 mM NaCl.
high ionic strength: -favors dissociation of protein–protein and
-protein–nucleic acid interactions.
-cause extreme stacking of proteins in the sample well resulting in
-protein aggregation, especially of membrane proteins.
Higher salt conc, are largely avoided during protein isolation e.g. 4500 mM.
2. pH
The pH of all native gels is kept in the neutral or slightly alkaline range which seems to be tolerated by most protein complexes.
Isolation of stable protein complexes by native electrophoresis
14. 3. Choice of detergent for membrane protein complexes
Mild neutral detergents like Triton X-100 or dodecylmaltoside are commonly used to solubilize membrane proteins and complexes
for chromatographic purifications and also for native electrophoresis, as exemplified in Fig. 1.
As a general rule, detergent/protein ratios around 2–3 g/g are chosen in order to avoid extensive delipidation and denaturation.
Water-soluble proteins and complexes:
No require detergents for solubility and
commonly do not even bind detergent so that
essentially no risk of denaturation of water soluble proteins by detergents exists.
less prone to protein aggregation compared to membrane proteins. Therefore,
mostly give rise to sharp bands in CNE compared to the diffuse and broadened bands of membrane proteins.
15. Generally CNE
If tricine/imidazole or tricine/ bis-tris buffers are used, is limited to separation of acidic proteins with pI 7
If an alternative glycine/tris-buffer is used expand the useful pH range to proteins with pI 8
This means that all stronger basic water soluble and membrane proteins are lost using CNE.
BNE and hrCNE
Enhance the solubility and anodic migration of membrane proteins, since hydrophobic proteins bind the anionic
Coomassie-dye (in BNE) or mixed anionic/neutral detergent micelles (in hrCNE)
many water-soluble proteins do not bind these anionic compounds and
therefore no improvement compared to CNE is observed for these proteins.
But many water-soluble proteins and complexes have been successfully separated by BNE.
Separations of acidic water-soluble proteins by CNE,
Coomassie-binding and basic water-soluble proteins by BNE, and
of basic water soluble proteins that were capable to bind anionic detergent micelles.
Presumably all separations of membrane protein complexes by BNE performed before 1998 are examples for the
separation/isolation of (detergent-) stable complexes, with the exception of a paper by Grandier-Vazeille and Guerin
that anticipated several features necessary for the isolation of supercomplexes.
Further milestones in the isolation of detergent-labile associations of membrane protein complexes were the isolation
of dimeric ATP synthase from yeast and respiratory supercomplexes from yeast and mammals.
16.
17.
18. Conservation of detergent-labile protein–protein interactions
few alternatives are currently available to preserve detergent-labile interactions in supramolecular structures of membrane protein
complexes:
(i) if common neutral detergents like Triton X-100/dodecylmaltoside are used, as described by BNE, the detergent/protein ratio must be
kept low and close to the solubilization-limits, as exemplified by the isolation of dimeric ATP synthase from yeast.
(ii) Neutral detergents milder than Triton X-100/dodecylmaltoside can be used, digitonin, as exemplified in Fig. 1.
The digitonin/protein ratio is less critical for the isolation of suprastructures.
Reducing the digitonin/protein ratio to near the solubilization-limits offers ideal conditions to isolate very large assemblies like
respiratory supercomplexes by BNE.
Another very mild detergent, BigCHAP, has recently been introduced to isolate oligomeric ATP synthase.
(iii) In addition to detergent effects on the stability of supramolecular structures, also the choice of the native electrophoresis variant can
have remarkable effects.
Separating the same sample by BNE and CNE revealed that respiratory supercomplexes from yeast mutants deficient in cardiolipin
synthase are stable in CN gels but dissociate into the individual complexes III and IV in BN gels.
The milder conditions in CNE compared to BNE have also been demonstrated recently for mammalian ATP synthase. However, since
membrane protein complexes often migrate as very broad bands in CN gels, BNE remains the method of choice to isolate more stable
supramolecular assemblies.
Dissociation of supercomplexes by detergents is partly explained by breaking of hydrophobic protein–protein interactions but also
lipids like cardiolipin in general and one specific cardiolipin molecule at the interface of respiratory complexes III and IV seem to be
important components to glue yeast respiratory complexes III and IV together into respirasomes.
minor modifications of cardiolipin structure, induced by mutations in the yeast and human taffazin genes, affect respirasome
stability in yeast and in patients suffering from the devastating Barth syndrome
19. 2-D native gels, native in both dimensions, reduce sample complexity and reveal loosely associated proteins
In principle, detergent-labile and non-permanently associated proteins identified by in vivo cross-linking prior to
protein solubilization by detergents and separation by BNE.
A simpler alternative to identify interacting/associated proteins is isolation of supramolecular assemblies under mildest
conditions, i.e. using low ionic strength, neutral pH, mildest detergents and mildest electrophoresis variants. However,
the complexity of the supramolecular structure may cause analytical problems if the sample contains too many
protein components or if the mass differences between subunits are too small.
Therefore, supramolecular assemblies from mammalian mitochondria separated by 1-D BNE containing around 70
subunits were dissociated into the individual complexes by modified BNE in a second native dimension gel, as shown
in Fig. 3H.
The properties of modified 2-D BNE harsher than of 1-D BNE to release individual complexes from supercomplexes.
This achieved by adding detergent (0.02% dodecylmaltoside) to the Coomassie containing cathode buffer.
Application of 2-D BN to proteomic analysis of mammalian ATP synthase revealed two novel associated proteins,
AGP and MLQ.
Recently, combination of 1-D BNE & 2-D hrCNE to 2-D BN/hrCN systems used to characterize low abundant
respiratory supercomplexes in the yeast Yarrowia lipolytica.
2-D native electrophoresis using the 2-D BN/hrCN combination was found ideal for fluorescent and functional assays
of mitochondrial complexes
20. Identification of interactions b/n highly hydrophobic proteins under SDS-PAGE conditions
Identification of protein–protein interactions under SDS-PAGE is restricted to highly hydrophobic proteins
few suitable proteins found empirically
These limitations seem not really encouraging but no useful alternative techniques are currently available.
Highly hydrophobic subunit c from mitochondrial ATP synthases is known to form oligomeric rings in the native
enzyme.
In some organisms, oligomeric rings resist unfolding by SDS so that
c10-rings observed in SDSgels provided that the applied sample is not heated in SDS solution.
Very recently, realized that c-rings from yeast ATP synthase remained almost quantitatively associated with
another neighboring hydrophobic protein, the a-subunit, in 2-D BN/SDS gels.
Further identification of a dimeric form of the c10a-complex (c10a)2 and of dimeric subunit a (a2) suggested that
the two c-rings in dimeric ATP synthase are linked together via subunit a-dimers.
The subunit c-a association is easily identified by a 2-D electrophoretic technique known as dSDS-PAGE as
exemplified in Fig.3I.
For this purpose, monomeric ATP synthase is isolated by 2-D BN/BN gels similar to Fig. 3H.
Individual spots, e.g. the ATP synthase (boxed red), are then cut out from the gel and separated by dSDS-PAGE,
i.e. by two special orthogonal SDS-gels
21. Development of techniques associated with BNE and CNE
The basic task of native electrophoresis is isolation of proteins and protein complexes on the
microgram scale for a number of general purposes.
These general tools have been applied in many specific research areas, for example,
to study protein import,
dynamics of proteasomes,
biogenesis and assembly of membrane protein complexes, and
to explore mitochondrial alterations in apoptosis, carcinogenesis, neurodegenerative disorders
and mitochondrial encephalomyopathies.
1. 1-D BN and 2-D BN/SDS gels and blots, and recovery of proteins from BN gels
2. Use of BNE to estimate mass and oligomeric state of native proteins, and to isolate highly pure
protein from chromatographically preenriched fractions
3. Protein quantification on 1-D BN and 2-D BN/ SDS gels for clinical applications
4. Amino-terminal sequencing of proteins on 2-D BN/SDS gels
5. Detection of fluorescence-labeled proteins on 2-D BN/SDS and 1-D hrCN gels
6. In-gel catalytic activity assays for clinical and biochemical research
7. Advanced structural investigations using protein extracts from BN gels
22. SDS PAGE Native PAGE
Description
SDS PAGE is a separation technique that separ
ates proteins on the basis of their mass.
Native PAGE is an electrophoretic technique t
hat separates proteins on the basis of their size
and charge.
Nature of Gel
The gel is denatured. The gel is not denatured.
Denaturation
SDS is added to the gel to impart a negative ch
arge on the protein samples.
No such activity is required.
Basis of Separation
The proteins are separated on the basis of mass
.
The proteins are separated on the basis of size
and charge.
Protein Stability and Recovery
The proteins are not stable in the SDS PAGE, a
nd hence cannot be recovered.
The proteins are stable in a Native PAGE, and
can be recovered later.
What are the applications of native PAGE?
Native PAGE is used for
•Characterization of soluble protein complexes, and
•Isolation of protein complexes from biological membranes.
Editor's Notes
-BN-PAGE is a charge shifted method, which employs the electrolytic mobility of protein complex as it migrates through a gel gradient
-protein intrinsic (PI)A protein (group of proteins) permanently attached to a biological membrane by interactions between its hydrophobic dimer units and the phospholipids of the membrane.
embedded in the lipid bilayer
-The isoelectric point (pI) is the pH of a solution at which the net charge of a protein becomes zero.
Furthermore, hydrophobic proteins with bound dye obtain solubility properties comparable to water soluble proteins.
No detergent is required to keep membrane proteins solubilized when they migrate through detergent-free gels.
CNE: cathode buffer for CNE does not contain detergent or dye. Proteins migrate according to their intrinsic charge in CNE.
BNE: dark blue cathode buffer for BNE containing 0.02% Coomassie dye can be replaced during electrophoresis by a similar buffer with tenfold lower dye content. This buffer exchange makes migrating protein bands visible during the electrophoretic run.
hrCNE1-3: mixed anionic micelles from neutral detergents (dodecylmaltoside or Triton X-100) and anionic detergent (deoxycholate) are added to the CNE buffer to induce a negative charge shift on proteins.
Buffer for hrCNE-3 is mildest. a) No pH correction except with tricine or with imidazole.
CNE: cathode buffer for CNE does not contain detergent or dye. Proteins migrate according to their intrinsic charge in CNE. neutral detergents (dodecylmaltoside or Triton X-100)
BNE: dark blue cathode buffer for BNE containing 0.02% Coomassie dye can be replaced during electrophoresis by a similar buffer with tenfold lower dye content.
This buffer exchange makes migrating protein bands visible during the electrophoretic run. neutral detergents (dodecylmaltoside or Triton X-100)
hrCNE1-3: mixed anionic micelles from neutral detergents (dodecylmaltoside or Triton X-100) and anionic detergent (deoxycholate) are added to the CNE buffer to induce a negative charge shift on proteins.
Buffer for hrCNE-3 is mildest. a) No pH correction except with tricine or with imidazole.
isolation of dimeric ATP synthase from yeast and respiratory supercomplexes from yeast and mammals.
isolation of dimeric ATP synthase from yeast and respiratory supercomplexes from yeast and mammals.