Your browser is too old
We can't provide a great video experience on old browserUpdate now
Cell Isolation Techniques
In a ﬂuorescence-activated cell sorter, a cell passing through the laser beam is monitored for ﬂuorescence. Droplets containing single cells are given a negative or positive charge, depending on whether the cell is ﬂuorescent or not. The droplets are then deﬂected by an electric field into collection tubes according to their charge. In the production of hybrid cells, it is possible to fuse one cell with another to form a heterokaryon, a combined cell with two separate nuclei. A heterokaryon proceeds to mitosis and produces a hybrid cell in which the two separate nuclear envelopes have been disassembled, allowing all the chromosomes to be brought together in a single large nucleus. In the preparative ultracentrifuge, the sample is contained in tubes that are inserted into a ring of angled cylindrical holes in a metal rotor. Some fractionation methods require a different type of rotor called a swinging-bucket rotor. In this case, the sample tubes are placed in metal tubes on hinges that allow the tubes to swing outward when the rotor spins. In the technique Cell fractionation by centrifugation, repeated centrifugation at progressively higher speeds will fractionate homogenates of cells into their components. In general, the smaller the subcellular component, the greater the centrifugal force required to sediment it. In velocity sedimentation, subcellular components sediment at different speeds according to their size and shape when layered over a solution containing sucrose. In equilibrium sedimentation, subcellular components move up or down when centrifuged in a gradient until they reach a position where their density matches their surroundings.
In the separation of molecules by column chromatography, the sample is applied to the top of a cylindrical glass or plastic column filled with a permeable solid matrix, such as cellulose. A large amount of solvent is then passed slowly through the column and collected in separate tubes as it emerges from the bottom. In ion exchange chromatography, the insoluble matrix carries ionic charges that retard the movement of molecules of opposite charge. Analogous matrices based on agarose or other polymers are also frequently used. In gel-filtration chromatography, the small beads that form the matrix are inert but porous. Affinity chromatography uses an insoluble matrix that is covalently linked to a specific ligand, such as an antibody molecule or an enzyme substrate, that will bind a specific protein. In Protein purification by chromatography, typical results obtained when three different chromatographic steps are used in succession to purify a protein. Proteins with the lowest affinity for the ion-exchange resin passed directly through the column and were collected in the earliest fractions eluted from the bottom of the column. The remaining proteins were eluted in sequence according to their affinity for the resin. The elution position of the still-impure protein was again determined by its enzymatic activity, and the active fractions were pooled and purified to homogeneity on an affinity column that contained an immobilized substrate of the enzyme.
In SDS polyacrylamide-gel electrophoresis, individual polypeptide chains form a complex with negatively charged molecules of sodium dodecyl sulfate (SDS) and therefore migrate as a negatively charged SDS–protein complex through a porous gel of polyacrylamide. Because the speed of migration under these conditions is greater the smaller the polypeptide, this technique can be used to determine the approximate molecular weight of a polypeptide chain as well as the subunit composition of a protein. In Separation of protein molecules by isoelectric focusing, at low pH (high H+ concentration), the carboxylic acid groups of proteins tend to be uncharged and their nitrogen containing basic groups fully charged, giving most proteins a net positive charge. At high pH, the carboxylic acid groups are negatively charged, and the basic groups tend to be uncharged, giving most proteins a net negative charge. At its isoelectric pH, a protein has no net charge since the positive and negative charges balance. Mass spectrometers used in biology contain an ion source that generates gaseous peptides or other molecules under conditions that render most molecules positively charged. The two major types of ion source are MALDI and electrospray. Ions are accelerated into a mass analyzer, which separates the ions on the basis of their mass and charge by one of three major methods:
- Time-of-ﬂight (TOF) analyzers
- Quadropolemass flters
- Ion traps
Tandem mass spectrometry typically involves two mass analyzers separated by a collision chamber containing an inert, high-energy gas.
The method ´´Measurement of binding with ﬂuorescence anisotropy´´ depends on a ﬂuorescently tagged protein that is illuminated with polarized light at the appropriate wavelength for excitation; a ﬂuorimeter is used to measure the intensity and polarization of the emitted light. If the ﬂuorescent protein is fixed in position and therefore does not rotate during the brief period between excitation and emission, then the emitted light will be polarized at the same angle as the excitation light. This directional effect is called ﬂuorescence anisotropy. In X-ray crystallography, a narrow beam of x-rays is directed at a well-ordered crystal. The atoms in the crystal scatter some of the beam, and the scattered waves reinforce one another at certain points and appear as a pattern of diffraction spots. This diffraction pattern, together with the amino acid sequence of the protein, can be used to produce an atomic model. Restriction nucleases cleave DNA at specific nucleotide sequences. Restriction enzymes often work as dimers, and the DNA sequence they recognize and cleave is often symmetrical around a central point. Both strands of the DNA double helix are cut at specific points within the target sequence. Some enzymes, such as HaeIII, produces blunt ends, while others, such as EcoRI and HindIII, produces sticky ends. DNA molecules can be separated by size using gel electrophoresis.
DNA Analysis and Manipulation
While the synthesis of cDNA, total mRNA is extracted from a particular tissue, and the enzyme reverse transcriptase is used to produce DNA copies (cDNA) of the mRNA molecules. A short oligonucleotide complementary to the poly-A tail at the 3ʹ end of the mRNA is first hybridized to the RNA to act as a primer for the reverse transcriptase, which then copies the RNA into a complementary DNA chain, thereby forming a DNA–RNA hybrid helix. PCR uses repeated rounds of strand separation, hybridization, and synthesis to amplify DNA. All the newly synthesized fragments serve as templates in their turn. Because the polymerase and the primers remain in the sample after the first cycle, PCR involves simply heating and then cooling the same sample, in the same test tube, again and again. PCR can be used to obtain either genomic or cDNA clones. To use PCR to clone a segment of chromosomal DNA, total genomic DNA is first purified from cells. Production of large amounts of a protein from a protein coding DNA sequence cloned into an expression vector and introduced into cells. A plasmid vector has been engineered to contain a highly active promoter, which causes unusually large amounts of mRNA to be produced from an adjacent protein-coding gene inserted into the plasmid vector. Depending on the characteristics of the cloning vector, the plasmid is introduced into bacterial, yeast, insect, or mammalian cells, where the inserted gene is efficiently transcribed and translated into protein.