Biochemistry

Dialysis is a common technique used in biochemistry for separating molecules based on diffusion. In this procedure, a semipermeable membrane allows the movement of certain molecules based on size. This method can be applied to the removal of buffer, known as desalting, or exchanging buffer molecules or ions from a protein solution. This video covers the principles of dialysis along with a general procedure.  Several applications of dialysis are reviewed, including the removal of gradient...

Enzyme kinetics describes the catalytic effects of enzymes, which are biomolecules that facilitate chemical reactions necessary for living organisms. Enzymes act on molecules, referred to as substrates, to form products. Enzyme kinetic parameters are determined via assays that directly or indirectly measure changes in substrate or product concentration over time.  This video will cover the basic principles of enzyme kinetics (including rate equations) and kinetic models. The concepts governing...

Matrix-assisted laser desorption ionization (MALDI) is a mass spectrometry ion source ideal for the analysis of biomolecules. Instead of ionizing compounds in the gaseous state, samples are embedded in a matrix, which is struck by a laser. The matrix absorbs the majority of the energy; some of this energy is then transferred to the sample, which ionizes as a result. Sample ions can then be identified using a time-of-flight analyzer (TOF). This video covers principles of MALDI-TOF, including...

In tandem mass spectrometry a biomolecule of interest is isolated from a biological sample, and then fragmented into multiple subunits in order to help elucidate its composition and sequence. This is accomplished by having mass spectrometers in series. The first spectrometer ionizes a sample and filter ions of a specific mass to charge ratio. Filtered ions are then fragmented and passed to a second mass spectrometer where the fragments are analyzed. This video introduces the principles of...

Protein crystallization, obtaining a solid lattice of biomolecules, elucidates protein structure and enables the study of protein function. Crystallization involves drying purified protein under a combination of many factors, including pH, temperature, ionic strength, and protein concentration. Once crystals are obtained, the protein structure can be elucidated by x-ray diffraction and computation of an electron density model. This video introduces protein crystallization and shows a general...

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase,...

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights. The procedure in this video covers the main...

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs that mimic the structure of natural biomolecules. Cells utilize analogs in their endogenous biochemical processes, producing compounds that are labeled. The label allows for the incorporation of detection and affinity tags, which can then be used to elucidate metabolic pathways using other biochemical analytical techniques, such as SDS-PAGE...

The electrophoretic mobility shift assay (EMSA) is a biochemical procedure used to elucidate binding between proteins and nucleic acids. In this assay a radiolabeled nucleic acid and test protein are mixed. Binding is determined via gel electrophoresis which separates components based on mass, charge, and conformation. This video shows the concepts of EMSA and a general procedure, including gel and protein preparation, binding, electrophoresis, and detection. Applications covered in this video...

Measuring the concentration is a fundamental step of many biochemical assays. Photometric protein determination takes advantage of the fact that the more a sample contains light-absorbing substances, the less the light will transmit through it. Since the relationship between concentration and absorption is linear, this phenomenon can be used to measure the concentration in samples where it is unknown. This video describes the basics of photometric protein determination and introduces the...

Density gradient ultracentrifugation is a common technique used to isolate and purify biomolecules and cell structures. This technique exploits the fact that, in suspension, particles that are more dense than the solvent will sediment, while those that are less dense will float. A high-speed ultracentrifuge is used to accelerate this process in order to separate biomolecules within a density gradient, which can be established by layering liquids of decreasing density in a centrifuge tube. The...

Co-immunoprecipitation (CoIP) and pull-down assays are closely related methods to identify stable protein-protein interactions. These methods are related to immunoprecipitation, a method for separating a target protein bound to an antibody from unbound proteins. In CoIP, an antibody-bound protein is itself bound to another protein that does not bind with the antibody, this is followed by a separation process that preserves the protein-protein complex. The difference in pull-down assays is that...

Reconstitution is the process of returning an isolated biomolecule to its original form or function. This is particularly useful for studying membrane proteins, which enable important cellular functions and affect the behavior of nearby lipids. To study the function of purified membrane proteins in situ, they must be reconstituted by integrating them into an artificial lipid membrane. This video introduces membrane protein reconstitution concepts and related procedures, such as protein...

Förster resonance energy transfer (FRET) is a phenomenon used to investigate close-range biochemical interactions. In FRET, a donor photoluminescent molecule can non-radiatively transfer energy to an acceptor molecule if their respective emission and absorbance spectra overlap. The amount of energy transferred—and consequently the overall emission of sample—depends on the proximity of an acceptor-donor pair of photoluminescent molecules. FRET analysis is combined with other biochemistry...

Surface plasmon resonance (SPR) is the underlying optical phenomenon behind label-free biosensors to evaluate the molecular affinity, kinetics, specificity, and concentration of biomolecules. In SPR, biomolecular interactions occur on a biosensor made of a thin layer of metal on a prism. Real-time interactions of biomolecules can be monitored by measuring the changes of light reflected off the underside of the metal. This video describes the basic concepts of SPR and how it is used to analyze...