9: Techniques
- Page ID
- 2930
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)The environment of a cell is very complex, making it very diffcult, if not impossible, to study individual reactions, enzymes, or pathways within it. For this reason, biochemists prefer to isolate molecules (enzymes, DNAs, RNAs, and other molecules of interest) so they can be analyzed without interference from the millions of other processes occurring simultaneously in the cell. Many of the methods used in isolating molecules from cells involve some form of chromatography. To separate compounds from their cellular environments, one must first break open (lyse) the cells. In this section, we describe some of the methods biochemists use to do their work.
- 9.1: Cell Disruption
- There are several ways to break open cells. Whatever method is employed, the crude lysates obtained contain all of the molecules in the cell, and thus, must be further processed to separate the molecules into smaller subsets, or fractions.
- 9.2: Fractionation
- Fractionation of samples typically starts with centrifugation. Using a centrifuge, one can remove cell debris, and fractionate organelles, and cytoplasm. For example, nuclei, being relatively large, can be spun down at fairly low speeds. Once nuclei have been sedimented, the remaining solution, or supernatant, can be centrifuged at higher speeds to obtain the smaller organelles, like mitochondria. Each of these fractions will contain a subset of the molecules in the cell.
- 9.3: Ion Exchange Chromatography
- In ion exchange chromatography, the support consists of tiny beads to which are attached chemicals possessing a charge. Each charged molecule has a counter-ion.
- 9.4: Gel Exclusion Chromatography
- Gel exclusion chromatography is a low resolution isolation method. This involves the use of beads that have tiny “tunnels" in them that each have a precise size. The size is referred to as an “exclusion limit," which means that molecules above a certain molecular weight will not fit into the tunnels. Molecules with sizes larger than the exclusion limit do not enter the tunnels and pass through the column relatively quickly by making their way between the beads.
- 9.5: Affinity Chromatography
- Affinity chromatography exploits the binding affinities of target molecules (typically proteins) for substances covalently linked to beads. For example, if one wanted to separate all of the proteins in a sample that bound to ATP from proteins that do not bind ATP, one could covalently link ATP to support beads and then pass the sample through column. All proteins that bind ATP will “stick" to the column, whereas those that do not bind ATP will pass quickly through it.
- 9.7: Histidine Tagging
- Histidine tagging is a powerful tool for isolating a recombinant protein from a cell lysate. The protein produced when this gene is expressed has a run of histidine residues fused at either the carboxyl or amino terminus to the amino acids in the remainder of the protein. The histidine side chains of this “tag" have an affinity for nickel or cobalt ions, making separation of histidine tagged proteins from a cell lysate is relatively easy.
- 9.8: Electrophoresis
- DNA molecules are long and loaded with negative charges, thanks to their phosphate backbones. Electrophoretic methods separate large molecules, such as DNA, RNA, and proteins based on their charge and size. For DNA and RNA, the charge of the nucleic acid is proportional to its size (length). For proteins, which do not have a uniform charge, a clever trick is employed to make them mimic nucleic acids.
- 9.10: Microarrays
- DNA microarrays, for example, can be used to determine all of the genes that are being expressed in a given tissue, simultaneously. Microarrays employ a grid (or array) made of rows and columns on a glass slide, with each box of the grid containing many copies of a specific molecule, say a single-stranded DNA molecule corresponding to the sequence of a single unique gene.