Chemistry: electrical and wave energy – Apparatus – Electrophoretic or electro-osmotic apparatus
Reexamination Certificate
2001-05-18
2003-11-25
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrophoretic or electro-osmotic apparatus
C204S462000, C204S466000, C204S616000, C204S467000, C204S618000, C422S091000
Reexamination Certificate
active
06652724
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to a method and apparatus for automatically identifying the location of a biological sample in an electrophoresis gel and transferring the sample to a sample plate. More particularly, the invention is directed to a computer assisted method and to a computer controlled apparatus for excising a gel spot from an electrophoresis gel slab and transferring the gel spot to a multiwell sample plate.
BACKGROUND OF THE INVENTION
Genomes provide the sequence information required to construct proteins that are the working parts of living cells. Genomes and genes are linear constructs composed of four different nucleotides arranged in triplet condons that specify the order and identity of the approximately 20 different amino acids that make up proteins. The nucleic acids are chemically very similar, and are arranged in very long contiguous sequences with intervening non-coding regions. For analysis, nucleic acids must be cut up into fragments of tractable length using shearing forces or restriction enzymes which cut the nucleic acid at specific known sites.
Proteins are made of amino acid subunits that have a range of different isoelectric points, molecular weights, and solubility or hydrophobicity characteristics. The synthesized peptides have exactly defined lengths, and roll up or are assembled into proteins of well defined molecular weights. The estimated 100,000 different primary proteins in man have a range of charge densities and isoelectric points, solubilities, and surface characteristics not found in nucleic acids. Further, proteins have a range of surface conformations which mediate specific interactions between proteins, between proteins and nucleic acids, and, in the form of enzymatically active sites, between low molecular weight metabolites, and all the various types of macromolecules found in cells and foodstuffs. Proteins are the molecular machines that carry out the panoply of syntheses, disassemblies and degradations, immunochemical defense reactions, and paratactic interactions that underlie the assembly of membranes and subcellular organelles.
There is a need for analytical methods that allow a large fraction of the total number of proteins present in a cell or tissue to be detected and quantitated. The quantitative analysis of large sets of proteins that have such a wide variety of functions, sizes, conformation, activities, solubilities, and charge characteristics is both a centrally important challenge, and an exceedingly difficult problem. The problem is rendered even more difficult by the requirement that analysis detecting thousands of proteins per analyses be done in parallel on relatively large numbers of samples in a reasonable time to do experimental toxicological and pharmacological studies.
The electrophoretic mobility of a non-denatured protein is a function of the surface charges of either the monomeric protein or the sum of the surface charges of the subunits, and these are generally used under rate-zonal conditions, i.e., under conditions where the proteins move through a gel or other support at one pH. The distance traveled is a function of the charge to mass ratio, and a function of electrophoresis time. Second dimension separations are done in gradient gels of decreasing pore size such that proteins move until movement essentially ceases as the protein reach pore sizes that prevent further movement. Experimental attempts to develop two dimensional methods based on these parameters using non-denaturing conditions have not yielded the resolution required.
Two-dimensional methods involving denaturing conditions have been explored and widely adopted. The initial separation is done in concentrated urea in the presence of ampholytes which are a heterogeneous mixture of synthetic polymers having wide variation in the ratio of acidic to basic groups. When these are subjected to an electrical field in a gel, the ampholytes sort themselves out into a continuous series based on the isoelectric point. Proteins move along the gel until they reach their own isoelectric point and stop. Further, since the proteins are denatured and unrolled, their isoelectric points reflect the sum of all of the charged groups in the protein, whether previously external or internal in the native state. The isoelectric point determination in such a separation can be calculated from the amino acid composition of the protein, and is a valuable parameter for protein classification.
The second dimensional separation is based on the length (and hence the mass) of the unrolled denatured protein and takes place in the following way. Proteins from the isoelectric separation are exposed to a highly charged detergent which has attached the longest paraffin chain which will remain extended in solution, and not fold back on itself. Sodium dodecyl sulfate (SDS) is the detergent of choice, and in solution will uniformly coat unrolled polypeptide chains, and attach to them by hydrophobic linkages, leaving the highly charged sulfate groups on the surface. The result is particles of approximately rod shape having approximately equal charge-to-mass ratios. Particles having equal charge-to-mass ratios move at the same rate in electrical fields, so that all proteins covered with SDS should have equal mobility in solution. However, if electrophoresis of such particles is done in a microporous gel, then larger particles will be retarded relative to smaller ones.
In practice, the resolutions of these two separate methods are quite high. At least 150 proteins can be resolved from a suitable mixture by isoelectric focusing, and an equal number resolved from a suitable protein mixture by SDS electrophoresis. If the two processes can be mated together in a two-dimensional array, the final resolution should be the product of the resolution of the two methods separately, i.e., 150
2
or 22,500. Experimentally, as many as 5,000 proteins have been resolved in large two-dimensional electrophoresis gels, and the theoretical resolution of current electrophoresis as calculated from spot sizes, and the number of spots which could theoretically be packed into the gel area used is around 30,000.
It is quite evident that a key step in the high-resolution two-dimensional electrophoresis technique using isoelectric focusing followed by SDS electrophoresis in the second dimension is mating the two methods together without the loss of resolution inherent in collecting and separately analyzing fractions.
Experimentally, isoelectric focusing is done under temperature controlled conditions in glass tubes (ISO tubes) having an internal diameter of approximately 0.5-2 mm, and approximately 30 cm long. ISO tubes are then attached to a small syringe full of water or buffer solution, and the gels extruded by hand along the top of a second-dimension gel cast between two glass plates. An empty space is typically formed between the top of the gel and the top of the plates. The gels are carefully extruded into this space by a double movement in which the syringe plunger is moved to extrude the gel as the ISO tube containing the gel is moved laterally along the top of the second dimension gel.
The molecules of the test sample migrate through the second dimension gel under the influence of an electric current to isolate the biomolecules. The gel is stained with various stains, such as silver or fluorescent compounds, to visualize the biomolecules. The stained biomolecules are then cut from gel and analyzed by various processes. Typically, the biomolecules are manually cut from the gel slab in the form of a gel spot. The gel spot is then placed in a suitable container. Typically, a single biological sample can be isolated into hundreds of biomolecules that must be manually cut from the gel slab. Accordingly, there is a continuing need in the industry for an improved method and apparatus for cutting samples from a gel slab.
SUMMARY OF THE INVENTION
The present invention is directed to an automated apparatus for separating a sample or gel spot from a second dimension electrophoresis ge
Anderson N. Leigh
Goodman Jack
Michael Samuel
Davis Garrett V.
Large Scale Proteomics Corporation
Quan Elizabeth
Robbins John C.
Warden Jill
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