Long-term alteration of dopaminergic neurotransmission is known to modulate the D2/D3 receptor expression in the brain. The modulation can occur as a response to pathological processes or pharmacological intervention. The receptor density can be monitored by in vivo positron emission tomography (PET) of [11C] raclopride. To obtain accurate measurements of receptor-ligand interaction, it is essential to estimate binding parameters at true (if transient) equilibrium of bound and unbound ligand quantities. We designed this study as a comparison of two quantitative approaches to transient equilibrium, the TRansient EquilibriuM BoLus Estimation (TREMBLE) method and the Transient Equilibrium Model (TEM) method, to determine binding parameters at transient equilibrium with bolus injection of the radioligand. The data demonstrates that TREMBLE unlike TEM identified the time at which equilibrium existed. TREMBLE revealed that equilibrium prevailed at one or more times after bolus injection and identified differences of receptor density among regions such as putamen and caudate nucleus. We demonstrated that TREMBLE is a quantitative approach suitable for the study of pathophysiological conditions of certain types of neurotransmission the brain.

Most analytical electrophoreses of proteins are achieved by separation in polyacrylamide gels under conditions that ensure dissociation of proteins into individual polypeptide subunits and minimize aggregation. Most commonly, the anionic detergent sodium dodecyl sulfate (SDS) is used in combination with a reducing agent (β-mercaptoethanol or dithiothreitol) and with heating to dissociate proteins before loading onto the gel. SDS binding denatures the polypeptides and imparts a negative charge that masks their intrinsic charge. The amount of SDS bound is generally sequence-independent and proportional to molecular weight; at saturation, approximately one SDS molecule is bound per two amino acids, or ∼1.4 g of SDS per gram of polypeptide. Therefore, the migration of SDS-polypeptide complexes in an electric field is proportional to the relative size of the polypeptide chain, and its molecular weight can be estimated by comparison to protein markers of known molecular weight. However, hydrophobicity, highly charged sequences, and certain posttranslational modifications such as glycosylation or phosphorylation may also influence migration. Thus, the apparent molecular weight of modified proteins does not always accurately reflect the mass of the polypeptide chain. This protocol describes preparation and running of SDS-PAGE gels, followed by staining to detect proteins using Coomassie Brilliant Blue. Finally, the stained SDS-PAGE gel may be scanned to an image or preserved by drying.

Isolating membrane proteins from their native cells while maintaining structural and functional integrity is challenging. Many detergents have been developed over the years that interact favorably with membrane proteins and mimic the physical properties of the lipid bilayer. Choosing the appropriate detergent is crucial for the successful extraction of a protein in its properly folded, active conformation.

Pichia pastoris is a methylotrophic yeast capable of metabolizing methanol as its sole carbon source. Growth in methanol-containing medium results in dramatic induction of genes in the alcohol oxidation pathway including alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD), and dihydroxyacetone synthase (DHAS). These proteins may comprise up to 30% of the biomass. Investigators have exploited these methanol-dependent genes to generate tightly regulated expression vectors. Most Pichia vectors use the strong and tightly regulated AOX1 promoter to drive heterologous protein expression. Obtaining integrated Pichia transformants requires more DNA than transformations into Saccharomyces cerevisiae, where the gene is expressed from episomal plasmids; however, transformants are extremely stable and can be stored for many years.

For expression of some proteins in Escherichia coli, export to the periplasmic space is preferred over conventional expression in the cytosol. Export can be accomplished by fusing the coding sequence to DNA encoding a signal peptide (e.g., using pET-22b), which is cleaved by the bacterial signal peptidase as the protein is exported into the space between the inner and outer membranes of E. coli This protocol uses osmotic shock to release polypeptides from the periplasm. Although not quantitative, it should provide preliminary information on the cellular location of signal peptide fusion proteins.

Many Escherichia coli expression vectors make use of the lac operon. In general, the lac operator (lacO) is located downstream from the promoter of the target gene, so that binding of the lac repressor blocks transcription initiation until lactose or the isopropyl-β-d-thiogalactopyranoside (IPTG) analog is added. The protocol given here is intended for use with IPTG-inducible vectors. l-Arabinose-inducible systems derived from the ara operon offer an alternative to expression systems based on the lac operon; guidance for their use is also provided.

Many variations of the original Coomassie Brilliant Blue staining procedure are in use. This protocol describes some selected variations on the standard procedure that give comparable and consistent staining results for proteins in the 20- to 200-kDa range.

The expression of foreign proteins at high levels in Escherichia coli often results in the formation of cytoplasmic granules or inclusion bodies composed of insoluble aggregates of the expressed protein. These inclusion bodies can be seen with a phase-contrast microscope and are readily separated from most soluble and membrane-bound bacterial proteins, as described in this protocol.

Hemoglobin III (HbIII) is one of the two oxygen reactive hemoproteins present in the bivalve, Lucina pectinata. The clam inhabits a sulfur-rich environment and HbIII is the only hemoprotein present in the system which does not yet have a structure described elsewhere. It is known that HbIII exists as a heterodimer with hemoglobin II (HbII) to generate the stable Oxy(HbII-HbIII) complex but it remains unknown if HbIII can form a homodimeric species. Here, a new chromatographic methodology to separate OxyHbIII from the HbII-HbIII dimer has been developed, employing a fast performance liquid chromatography and ionic exchange chromatography column. The nature of OxyHbIII in solution at concentrations from 1.6 mg/mL to 20.4 mg/mL was studied using small angle X-ray scattering (SAXS). The results show that at all concentrations, the Oxy(HbIII-HbIII) dimer dominates in solution. However, as the concentration increases to nonphysiological values, 20.4 mg/mL, HbIII forms a 30% tetrameric fraction. Thus, there is a direct relationship between the Oxy(HbIII-HbIII) oligomeric form and hemoglobin concentration. We suggest it is likely that the OxyHbIII dimer contributes to active oxygen transport in tissues of L pectinata, where the Oxy(HbII-HbIII) complex is not present.

Recovery of intracellular proteins requires disruption of the host cell before the target protein is extracted and isolated. For cells enveloped in cell walls (such as Escherichia coli), vigorous methods are often required. This protocol focuses on E. coli lysis by sonication. Also included are methods for lysis by freeze-thaw and enzymatic treatments.

In immunoblotting (western blotting), proteins are first separated by SDS-PAGE and then transferred electrophoretically from the gel onto a support membrane that binds proteins tightly. After the unreacted binding sites of the membrane are blocked to suppress nonspecific adsorption of antibodies, the immobilized proteins are reacted with a specific polyclonal or monoclonal antibody. Antigen-antibody complexes are visualized using chromogenic, fluorescent, or chemiluminescent reactions. Immunoblotting protocols are reagent specific and, owing to the wide assortment of equipment, reagents, and antibodies available, highly diverse. Presented here is an example of a workable protocol for developing a blot using horseradish peroxidase (HRP)-conjugated secondary antibody and enhanced chemiluminescence (ECL). ECL is based on the emission of light during the HRP-catalyzed oxidation of luminal or other substrates. Emitted light is captured on film or by a CCD camera, for qualitative or semiquantitative analysis. Because ECL is so sensitive, it has become a popular detection method. This protocol can be modified for different membranes, antibodies, and detection systems. Optimal dilutions of the primary and secondary antibodies need to be determined empirically, but recommendations provided by the manufacturer are usually a good starting point.

Recovery of intracellular proteins requires disruption of the host cell before the target protein is extracted and isolated. Disruption methods vary depending on the type of cells, the total volume, and the number of samples being processed. For cells enveloped in cell walls (such as yeast), mild techniques such as hypotonic shock are not sufficient to achieve adequate lysis. More vigorous methods are often required. Although the preferred medium- or large-scale method of breaking yeast cells is mechanical shearing, lysis with the aid of glass beads in a BeadBeater is described here.

This protocol describes silver staining procedures to detect low-abundance proteins in sodium dodecyl sulfate-polyacrylamide gels.

Obtaining high quantities of a specific protein directly from native sources is often challenging, particularly when dealing with human proteins. To overcome this obstacle, many researchers take advantage of heterologous expression systems by cloning genes into artificial vectors designed to operate within easily cultured cells, such as Escherichia coli, Pichia pastoris (yeast), and several varieties of insect and mammalian cells. Heterologous expression systems also allow for easy modification of the protein to optimize expression, mutational analysis of specific sites within the protein and facilitate their purification with engineered affinity tags. Some degree of purification of the target protein is usually required for functional analysis. Purification to near homogeneity is essential for characterization of protein structure by X-ray crystallography or nuclear magnetic resonance (NMR) and characterization of the biochemical and biophysical properties of a protein, because contaminating proteins almost always adversely affect the results. Methods for producing and purifying proteins in several different expression platforms and using a variety of vectors are introduced here.

Determining the concentration of protein samples generally is accomplished either by measuring the UV absorbance at 280 nm or by reacting the protein quantitatively with dyes and/or metal ions (Bradford, Lowry, or BCA assays). For purified proteins, UV absorbance remains the most popular method because it is fast, convenient, and reproducible; it does not consume the protein; and it requires no additional reagents, standards, or incubations. No method of protein concentration determination is perfect because each is subject to a different set of constraints such as interference of buffer components and contaminating proteins in direct UV determination (A 280) or reactivity of individual proteins and buffer components with the detecting reagents in colorimetric assays. In cases in which protein concentration is critical (e.g., determination of catalytic rate constants for an enzyme), it may be advisable to compare the results of several assays.

Fusion proteins that contain a glutathione S-transferase (GST) moiety can be purified to near homogeneity by affinity chromatography on glutathione-linked resins. Glutathione immobilized on a chromatography matrix, such as agarose or Sepharose, acts as a substrate for the GST moiety of fusion proteins. Contaminating proteins are washed away, and the bound GST fusion proteins are then readily displaced from the resin by elution with buffers containing free glutathione.

The general strategy of the baculovirus expression system is to infect insect cells with a virus that expresses a foreign protein at a very late stage of infection. Almost all baculovirus expression systems use the procedures for insect cell transfection, baculovirus production, and protein expression given in the main portion of this protocol. This protocol also includes a method that uses molecular biology techniques to produce recombinant baculovirus DNA in E. coli before transfection of insect cells. It is important to quantify the viral titer to achieve optimal and reproducible expression of target proteins. Accordingly, the viral plaque assay is also described here.

The Bradford assay is a quick and fairly sensitive method for measuring the concentrations of proteins. It is based on the shift in absorbance maximum of Coomassie Brilliant Blue G-250 dye from 465 to 595 nm following binding to denatured proteins in solution.

Immobilized metal affinity chromatography (IMAC) is based on the affinity of polyhistidine tracts for divalent metal cations (usually Ni2+) immobilized as transition metal chelate complexes on a chromatography resin. The main protocol here is optimized for use of Ni2+-NTA resin to purify soluble 6xHis-tagged proteins by a straightforward batch method during the binding step, followed by gravity flow for washes and elution. This protocol does not require any specialized equipment other than a simple glass or plastic column. IMAC resins can be used in multiple formats, including batch, gravity flow, centrifuge columns, and fast performance liquid chromatography (FPLC) systems. FPLC systems are designed specifically for the chromatographic separations of proteins and other biomolecules. These systems typically contain multiple pumps, an in-line UV absorption monitor, conductivity meter, pH meter, fraction collector, and other options that allow for the simultaneous purification, analysis, and fractionation of the sample. When linked with the appropriate instruments, an FPLC can become a high-precision, automated instrument that separates proteins at a high resolution. An alternative protocol is included here that describes 6xHis-tagged protein purification using FPLC. Procedures for the cleaning and regeneration of the IMAC resin for reuse are also described, and, finally, considerations for storing purified proteins are discussed.

The pre-mRNA branch point sequence (BPS) anneals with a pseudouridine-modified region of the U2 small nuclear (sn)RNA, and offers a 2' hydroxyl group of a bulged adenosine as the nucleophile for the first catalytic step of pre-mRNA splicing. To increase our structural understanding of branch site selection, we characterized a duplex containing a BPS sequence that is common among multicellular eukaryotes (5'-UACUGAC-3') and the complementary U2 snRNA site using NMR. A major conformation of the expected branch site adenosine stacked within the duplex and paired with the conserved pseudouridine of the U2 snRNA strand. In contrast, the guanosine preceding the branch site appeared flexible and had weak contacts with the surrounding nucleotides. Pseudouridine-modified and unmodified U2 snRNA-BPS-containing duplexes remained structurally similar. These results highlight the importance of auxiliary factors to achieve the active bulged conformation of the branch site nucleophile for the first step of pre-mRNA splicing.