Electrophoresis Articles
Proteins can also be detected immunologically following electrophoresis, a technique known as Western blotting. This method relies on the fact that most epitopes (sites recognized by antibodies, generally comprising several amino acids) are still recognizable…
One of the most straightforward applications of immunological detection is the ELISA or enzyme-linked immunosorbent assay. In the simplest system, the bound antigen is probed with antibodies that carry covalently attached enzyme molecules. Antibody binding…
Alkaline phosphatase catalyzes the removal of a phosphate group from its substrate. A variety of synthetic substrates have been constructed which, on phosphate hydrolysis, liberate chromogens or luminescent compounds. A commonly used chromogenic substrate is…
The highly specific binding interaction between antibodies and their unique antigens has been exploited to create sensitive and specific detection systems for proteins. An antibody can be raised and/or purified “against” (i.e. binding to) a…
Immunological detection of proteins requires that proteins be transferred and immobilized onto a membrane support after electrophoresis (see Western Blotting). Staining of the immobilized proteins establishes transfer efficiency, and allows the operator to mark the membrane…
In certain instances, the effects of staining a protein may interfere with subsequent analysis. Examples are Coomassie staining when enzymatic activity is required, or silver staining prior to amino acid analysis when covalent modification of…
Utilizing the same chemistry as black and white photography, silver staining is another highly sensitive method for the visualization of protein bands on electrophoresis gels. Silver ions are reduced to insoluble silver metal granules in…
The Coomassie dyes (R-250 and G-250) bind to proteins through ionic interactions between dye sulfonic acid groups and positive protein amine groups as well as through Van der Waals attractions. Coomassie R-250, the more commonly…
Fixing (or fixation) is the process whereby proteins are denatured and precipitated in large insoluble aggregates within the gel matrix. Fixation accomplishes several goals. Primarily, fixation prevents the diffusion of proteins, thus keeping the protein…
Autoradiographic Enhancement with Autofluor: The Procedure National Diagnostics’ Autofluor is an extremely sensitive, water based fluorographic enhancer for autoradiography on gels, TLC plates or paper chromatograms. GELS After staining, fix the gel with 5% glacial…
- Using PAGE to Determine Nucleic Acid Molecular Weight
- SSCP Analysis
- Sanger Sequencing
- Sample Preparation for Native PAGE of DNA
- Sample Prep for Denaturing PAGE of DNA
- S1 Mapping
- Run Conditions in Denaturing PAGE
- RNA Mapping
- RNA Electrophoresis
- Ribonuclease Protection
- Restriction Digest Mapping
- Primer Extension
- Preparing Denaturing DNA & RNA Gels
- Preparation of Denaturing Agarose Gels
- Preparation of Agarose Gels
- Pouring Sequencing Gels
- PFGE and FIGE
- PCR Analysis: Yield and Kinetics
- PCR Analysis: An Examination
- Native PAGE of DNA
- Mobility Shift Assay
- Methylation & Uracil Interference Assays
- Maxam & Gilbert Sequencing
- Manual Sequencing
- In Gel Enzyme Reactions
- Heteroduplex Analysis
- Gel Preparation for Native PAGE of DNA
- Gel Electrophoresis of PCR Products
- DNase I Footprinting
- DNA/RNA Purification from PAGE Gels
- DNA/RNA Purification from Agarose Gels – Electroelution
- Differential Display
- Denaturing Polyacrylamide Gel Electrophoresis of DNA & RNA
- Conformational Analysis
- Automated Sequencers
- Analysis of DNA/Protein Interactions
- Agarose Gel Electrophoresis of DNA and RNA – Uses and Variations
- Agarose Gel Electrophoresis of DNA and RNA – An Introduction