Electron microscopic immunocytochemistry. Silver enhancement of colloidal gold marker allows double labeling with the same primary antibody

Electron microscopic sections, immunocytochemically labeled with colloidal gold, can be prepared for double labeling by applying the "EM-silver enhancement" procedure. This method, a photographic, so-called physical, development, increases the size of the gold marker to a predeterminable value and thereby inactivates the anti-species antibody present on the gold grain, thus allowing the labeling of a second antigen with antibody raised in the same species.     

An alternative nonradioactive method for labeling DNA using biotin

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.     

Site-specific independent double labeling of proteins with reporter atoms.

Many types of physical, spectroscopic, and biological studies of proteins and other macromolecules are facilitated by the incorporation of reporter groups. In many cases these are single atom substitutes, for example isotopes (13C for C), or light (F for H) and heavy (Se for S) atom homologs. In some circumstances the incorporation of two different labels in the same molecule would be greatly desirable. Commonly used protein engineering methods for incorporating them can rarely cope with differential double labeling, and have other limitations such as universal, non-specific, or random incorporation. Although de novo peptide synthesis has the power to achieve highly specific labeling, the difficulties inherent in creating long sequences lead us to propose protein semisynthesis as the most practical approach. By ligating combinations of natural and labeled synthetic fragments to reform holoproteins, we can overcome any of the limitations discussed. Using cytochrome c as a model protein we show that two reporter atoms, selenium and bromine, can be simultaneously and site-specifically incorporated without significant consequences to structure and (or) function. This capability opens up the prospect of advances in a number of areas in structural biology.     

Flow cytometric double labeling technique for screening of multidrug resistance.

We investigated the capabilities of flow cytometry in the analysis of a multidrug resistant (MDR) human ovarian cancer cell line 2780AD and its drug sensitive parental A2780. A functional assay using daunorubicin (DNR) as a fluorescent probe was combined with an immunofluorescence assay of P-glycoprotein (P-gp) using the monoclonal antibody MRK-16. Functionally MDR could be demonstrated by the lower DNR-content of MDR cells compared to DNR-content of drug sensitive cells. When incubation was performed with DNR in the presence of verapamil, DNR-content increased in the MDR cells. However the content of the A2780 cells was never attained. Differences in DNR-content were not related to differences in DNA-content. In experimental cell lines immunofluorescence data were inversely related with those of DNR-content: MDR cells had high levels of P-gp expression and low levels of DNR-content (and vice versa in drug sensitive cells). Both assays can be easily combined in a multiparametric flow cytometric procedure to evaluate both parameters simultaneously in the same cells. Analysis of clinical samples demonstrates the existence of aberrant subpopulations which would not be detected by using a single parameter assay.     

Selenomethionine and selenocysteine double labeling strategy for crystallographic phasing

A protocol for the quantitative incorporation of both selenomethionine and selenocysteine into recombinant proteins overexpressed in Escherichia coli is described. This methodology is based on the use of a suitable cysteine auxotrophic strain and a minimal medium supplemented with selenium-labeled methionine and cysteine. The proteins chosen for these studies are the cathelin-like motif of protegrin-3 and a nucleoside-diphosphate kinase. Analysis of the purified proteins by electrospray mass spectrometry and X-ray crystallography revealed that both cysteine and methionine residues were isomorphously replaced by selenocysteine and selenomethionine. Moreover, selenocysteines allowed the formation of unstrained and stable diselenide bridges in place of the canonical disulfide bonds. In addition, we showed that NDP kinase contains a selenocysteine adduct on Cys122. This novel selenium double-labeling method is proposed as a general approach to increase the efficiency of the MAD technique used for phase determination in protein crystallography.     

A fluorescent probe which allows highly specific thiol labeling at low pH

Determination of the thiol-disulfide status in biological systems is challenging as redox pools are easily perturbed during sample preparation. This is particularly pertinent under neutral to mildly alkaline conditions typically required for alkylation of thiols. Here we describe the synthesis and properties of a thiol-specific reagent, fluorescent cyclic activated disulfide (FCAD), which includes the fluorescein moiety as fluorophore and utilizes a variation of thiol-disulfide exchange chemistry. The leaving-group character of FCAD makes it reactive at pH 3, allowing modification at low pH, limiting thiol-disulfide exchange. Different applications are demonstrated including picomolar thiol detection, determination of redox potentials, and in-gel detection of labeled proteins.     

ER-based double iCre fusion protein allows partial recombination in forebrain

Here we describe the generation of a new tamoxifen-inducible double Cre fusion protein generated by fusing two ERT2 domains onto both ends of the iCre recombinase (a codon improved Cre recombinase). This Cre fusion protein (ERiCreER) had a twofold increased activity in cell culture assays than the previously described MerCreMer Cre double fusion protein. ERiCreER was targeted to the brain by placing it under the control of the promoter from the CamKIIalpha gene using a 170 kb BAC. The fusion protein was detected in hippocampus, cortex, striatum, thalamus, and hypothalamus but not in cerebellum. The ERiCreER was cytoplasmatic in the absence of tamoxifen and translocated into the nucleus upon tamoxifen administration. The activity of the ERiCreER was tested in vivo by mating the CamKIIalpha ERiCreER transgenic line with mice harbouring exon 10 of the CREB gene flanked by two LoxP sites. In the absence of tamoxifen, no background activity was detected in mice older than 6 months. After tamoxifen administration, most if not all of the ERiCreER fusion protein translocated from the cytoplasm to the nucleus; however, only 5-10% of the "floxed" CREB allele was recombined. Recombination was also visualised at the cellular level by following the upregulation of the CREM protein, which corresponds precisely with CREB loss/recombination. Unlike in other tissues (Sohal et al., 2001; Tannour-Louet et al., 2002), it appears that in brain, although ERiCreER can bind tamoxifen, the Cre-recombinase cannot be fully activated.     

Analysis of data from experiments using double labeling

Frequently as a result of experiments in which two isotopes are used one is left with a sequence of samples, the ratio of labeling in each sample, and the problem of analyzing the ratios. Suppose that the experiments are designed so that one expects uniform labeling except for one or two special groups of samples. The problem, then, is to find these groups. Because of the variability in the count rate from sample to sample, the variance of the ratios differs from sample to sample making statistical analysis difficult. Furthermore, there is significant serial correlation in the sample disintegrations per minute for each of the isotopes. We have found that the serial correlation in the labeling ratio is small and of questionable significance in controls but becomes significant when there is a subsequence of samples in which the labeling ratio differs from that in the remainder of the gel. We examine the analysis of variance as a test for significant deviations in the labeling ratio and suggest a method for plotting deviations of labeling ratio from the average background labeling ratio. Finally, we develop a method of estimating the mean labeling ratio from the regression of disintegrations per minute of one isotope on those of the other isotope. This provides another way of plotting deviations in labeling ratio in terms of the residuals around the line of regression.     

Avidin-biotin-immunoglucose oxidase: use in single and double labeling procedures

We have investigated the use of an avidin-biotin-immunoglucose oxidase (AB-GO) technique for single and double antigen localization in conjunction with the avidin-biotin-immunoperoxidase (AB-P) technique in fixed, embedded specimens, using sequential monoclonal and polyclonal antibodies of the same species. The optimal technique for double labeling requires the first antibody to be applied and localized with the AB-P technique using 3,3'-diaminobenzidine (DAB) as the chromogen, followed by an optional elution step and/or incubation with mild detergent (0.01% Triton). The second antigen is localized with the AB-GO technique with nitro blue tetrazolium (NBT) as a chromogen. Effects of antigen concentration, intermediate elution steps, and the relative efficiency of the two methodologies are described.     

A novel procedure for pre-embedding double immunogold-silver labeling at the ultrastructural level.

Pre-embedding double immunogold-silver labeling using two ultrasmall gold conjugates has not been attempted previously because a means of distinguishing labels by conjugates of identical sizes was lacking. This study investigated the feasibility of creating a particle size segregation between two ultrasmall gold conjugates through sequential immunogold incubations and silver enhancements. Two primary antibodies, mouse anti-synaptophysin and rabbit anti-glial fibrillary acidic protein (GFAP), were used in the model system. Differentiation of the double labeling was achieved by incubating with one ultrasmall gold conjugate, followed by silver enhancement, and then incubating with the second ultrasmall gold conjugate, followed by additional silver enhancement. This resulted in two groups of silver-enhanced particles: smaller particles enhanced once and larger particles enhanced twice. Electron microscopic examination revealed two readily distinguished populations of gold-silver particles within the appropriate structures, with very little size overlap. The quality of the ultrastructure permitted identification of most subcellular organelles. This procedure provides for the first time a pre-embedding immunogold-silver labeling protocol that allows the precise subcellular co-localization of multiple antigens.     

Estimation of cell cycle parameters from double labeling experiments

Double labeling of cell populations with radioactive thymidine yields two types of differently labeled nuclei. Their numbers and the number of unlabeled nuclei can be used to estimate doubling times, T, and S-phase lengths, S. As of yet, such estimations have been performed either for stationary populations in which proliferation and losses are in balance, or for exponentially growing populations in which all cells have the same cycle duration. We calculate S and T for the more general type of cell population with arbitrarily distributed frequencies of cycle durations. The calculations do not require more mathematical or computational effort. We obtain three main results: (i) The estimation of T and S does not require explicit knowledge of the frequency distribution of cycle durations; (ii) in particular, equivalent estimates for T and S are obtained for both types of growing cell populations without losses, one with arbitrarily distributed cycle durations and one with the same cycle duration for all cells; and (iii) for small labeling indices, the estimate for S from the general model approaches the S-phase length of a stationary population and the estimate for T from the general model approaches the generation time of a stationary population, multiplied by the constant factor 1n(2). These relationships are valuable tools for reinterpreting results derived under the assumption of stationarity, which are considerably easier to obtain.     

Reversible labeling of cysteine-containing peptides allows their specific chromatographic isolation for non-gel proteome studies

We report upon a novel procedure to specifically isolate cysteine-containing peptides from a complex peptide mixture. Cysteines are converted to hydrophobic residues by mixed disulfide formation with Ellman's reagent. Proteins are subsequently digested with trypsin and the generated peptide mixture is a first time fractionated by reverse-phase high-performance liquid chromatography. Cysteinyl-peptides are isolated out of each primary fraction by a reduction step followed by a secondary peptide separation on the same column, performed under identical conditions as for the primary separation. The reducing agent removes the covalently attached group from the cysteine side chain, making cysteine-peptides more hydrophilic and, thereby, such peptides can be specifically collected during the secondary separation and are finally used to identify their precursor proteins using automated liquid chromatography tandem mass spectrometry. We show that this procedure efficiently isolates cysteine-peptides, making the sample mixture less complex for further analysis. This method was applied for the analysis of the proteomes of human platelets and enriched human plasma. In both proteomes, a significant number of low abundance proteins were identified next to extremely abundant ones. A dynamic range for protein identification spanning 4-5 orders of magnitude is demonstrated.     

Interaction of antigenic peptides with MHC class I molecules on living cells studied by photoaffinity labeling.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of antigenic peptides with murine MHC class I molecules on living cells. Photoreactive derivatives were prepared by N-terminal amidation with iodo, 4-azido salicylic acid of the Kd restricted Plasmodium berghei circumsporozoite (P.b. CS) peptide 253-260 (YIPSAEKI) and the Db-restricted Adenovirus 5 early region 1A (Ad5 E1A) peptide 234-243 (SGPSNTPPEI). As assessed in functional competition experiments, both peptide derivatives retained the specific binding activity of the parental peptides for Kd or Dd, respectively. The P.b. CS photoprobe specifically labeled Kd molecules on P815 (H-2d) cells, but failed to label RMA (H-2b) cells. Conversely, the Ad5 E1A photoprobe specifically labeled Db molecules on RMA cells, but failed to label P815 cells. When the two photoprobes were tested on a panel of Con A-activated spleen cells expressing 10 different H-2 haplotypes, significant photoaffinity labeling was observed only on H-2d cells with the P.b. CS photoprobe and on H-2b cells with the Ad5 E1A photoprobe. Labeling of cell-associated Kd or Db molecules with the photoprobes was specifically inhibited by antigenic peptides known to be presented by the same class I molecule. Photoaffinity labeling of Kd with the P.b. CS photoprobe was used to study the dynamics of peptide binding on living P815 cells. Binding increased steadily with the incubation period (up to 8 h) at 37 degrees C and at ambient temperature, but was greatly reduced (greater than 95%) at 0 to 4 degrees C or in the presence of ATP synthesis inhibitors. The magnitude of the labeling was twofold higher at room temperature than at 37 degrees C. In contrast, binding to isolated Kd molecules in solution rapidly reached maximal binding, particularly at 37 degrees C. Dissociation of the photoprobe from either cell-associated or soluble Kd molecules was similar, with a half time of approximately 1 h at 37 degrees C, whereas the complexes were long-lived at 4 degrees C in both instances.     

Dual labeling of a binding protein allows for specific fluorescence detection of native protein

Up-Converting Phosphor Technology (UPT) is based on lanthanide-containing, submicrometer-sized, ceramic particles that can absorb infrared light and emit visible light. Biological matrices do not up-convert; hence, there is no contribution to test background from sample autofluorescence. Up-converting phosphors do not photobleach and are inert to common assay interferants such as hemoglobin. A reader called UPlink has been developed to interrogate lateral flow test strips that utilize UPT labels. The reader contains a miniaturized, 1-W, infrared laser with peak emission at 980 nm. Preliminary assays that use up-converting phosphor labels, including tests for a drugs of abuse panel and Escherichia coli O157:H7, have been developed. In a "sandwich" assay format, 10(3) org/mL E. coli O157:H7 organisms were detectable in a negative control background of 10(9) other organisms per milliliter of culture medium. Coefficients of variation in concentrations tested from 0 to 10(7) org/mL were all < or =10%. In a competitive inhibition assay format, a multiplexed test simultaneously detected amphetamine, methamphetamine, phencyclidine, and opiates in saliva. For all assays, the percent displacement at 10 ng/mL was > or =40% demonstrating performance comparable with lab-based, commercially available EIAs. All assays were complete in 10 min. The development of rapid tests using UPT creates new applications for on-site testing with sensitivity not available using other label technologies.     

Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay

Standard microarrays measure mRNA abundance, not mRNA synthesis, and therefore cannot identify the mechanisms that regulate gene expression. We have developed a method to overcome this limitation by using the salvage enzyme uracil phosphoribosyltransferase (UPRT) from the protozoan Toxoplasma gondii. T. gondii UPRT has been well characterized because of its application in monitoring parasite growth: mammals lack this enzyme activity and thus only the parasite incorporates (3)H-uracil into its nucleic acids. In this study we used RNA labeling by UPRT to determine the roles of mRNA synthesis and decay in the control of gene expression during T. gondii asexual development. We also used this approach to specifically label parasite RNA during a mouse infection and to incorporate thio-substituted uridines into the RNA of human cells engineered to express T. gondii UPRT, indicating that engineered UPRT expression will allow cell-specific analysis of gene expression in organisms other than T. gondii.