Antigen was identified histochemically without the use of labeled antibodies by the sequential application of (a) specific rabbit antiserum, (b) sheep antiserum to rabbit immunoglobulin G, (c) specifically purified, soluble horseradish peroxidase-anti-horseradish peroxidase complex (PAP), (d) 3,3'-diaminobenzidine and hydrogen peroxide and (e) osmium tetroxide. A simple method for preparation of high yields of PAP consisted of precipitation of antibody from specific rabbit antiserum with horseradish peroxidase (PO) at equivalence, solubilization of the washed precipitate with excess PO at pH 2.3, 1°C, followed by immediate neutralization and separation of PAP from PO by half-saturation with ammonium sulfate. The ratio of PO to anti-PO in PAP was 3:2 irrespective of the source of antiserum. PAP was heterogeneous on electrophoresis, homogeneous on sedimentation, diffusion and electron microscopy and consisted of pentagons with diameters of 205 A. s20,w, 11.98 x 10–13; d20,w, 2.48 x 10–7; molecular weight by...

The unlabeled antibody enzyme method of immunohistochemistry preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes:

Muscle structure and theories of contraction.

▪ Abstract Eukaryotic translation initiation factor 4F (eIF4F) is a protein complex that mediates recruitment of ribosomes to mRNA. This event is the rate-limiting step for translation under most circumstances and a primary target for translational control. Functions of the constituent proteins of eIF4F include recognition of the mRNA 5′ cap structure (eIF4E), delivery of an RNA helicase to the 5′ region (eIF4A), bridging of the mRNA and the ribosome (eIF4G), and circularization of the mRNA via interaction with poly(A)-binding protein (eIF4G). eIF4 activity is regulated by transcription, phosphorylation, inhibitory proteins, and proteolytic cleavage. Extracellular stimuli evoke changes in phosphorylation that influence eIF4F activity, especially through the phosphoinositide 3-kinase (PI3K) and Ras signaling pathways. Viral infection and cellular stresses also affect eIF4F function. The recent determination of the structure of eIF4E at atomic resolution has provided insight about how translation is initiat...

eIF4 Initiation Factors: Effectors of mRNA Recruitment to Ribosomes and Regulators of Translation

An important part of characterizing any protein molecule is to determine its size and shape. Sedimentation and gel filtration are hydrodynamic techniques that can be used for this medium resolution structural analysis. This review collects a number of simple calculations that are useful for thinking about protein structure at the nanometer level. Readers are reminded that the Perrin equation is generally not a valid approach to determine the shape of proteins. Instead, a simple guideline is presented, based on the measured sedimentation coefficient and a calculated maximum S, to estimate if a protein is globular or elongated. It is recalled that a gel filtration column fractionates proteins on the basis of their Stokes radius, not molecular weight. The molecular weight can be determined by combining gradient sedimentation and gel filtration, techniques available in most biochemistry laboratories, as originally proposed by Siegel and Monte. Finally, rotary shadowing and negative stain electron microscopy are powerful techniques for resolving the size and shape of single protein molecules and complexes at the nanometer level. A combination of hydrodynamics and electron microscopy is especially powerful.

/pdf/size-and-shape-of-protein-molecules-at-the-nanometer-level-152mfw62l2.pdf

Size and Shape of Protein Molecules at the Nanometer Level Determined by Sedimentation, Gel Filtration, and Electron Microscopy

A negative staining method for high resolution electron microscopy of viruses.

Electron microscope investigations of the structure of collagen

Methods are described for determining the relative mass of particles in electron microscope specimens through the measurement of photographic densities in recorded images. These methods were applied to a quantitative study of the amounts of electron stains that could be associated with the particles of tomato bushy stunt virus (BSV) and tobacco mosaic virus (TMV). In the pH range above 2 where the viruses are stable, the amount of stain absorbed is too small to produce adequate contrast in the electron microscope. Maximum stain absorption was achieved at pH about 1 where with several reagents and combinations of reagents the mass of BSV could be increased to about four times that of the unstained particles. Optimum results were obtained with phosphotungstic acid alone or in combination with Pt, Th, or La ions. Since the pH conditions for high stain absorption are normally destructive, morphology is satisfactorily preserved only when the phosphotungstic acid is applied in concentrations of 10 per cent or greater or when the use of destructive reagents is preceded by a preliminary fixation under mild conditions. Maximum staining of TMV increased the mass of the particles to about two times that of the unstained. Estimates of the mass of heavily stained BSV particles indicate that their density is 3.3 gm./cm.(3) The high internal hydration of BSV probably accounts for the greater stain absorption and penetration compared to those of TMV which has very low or zero internal hydration. Anomalous images resulting from the use of electron stains are shown and discussed.

/pdf/electron-densitometry-of-stained-virus-particles-5glo7mvllb.pdf

Electron densitometry of stained virus particles.

1. Electron micrographs were made of myofibrils isolated from frog and other skeletal muscles fixed in formalin. The structure with respect to the location and disposition of the principal cross striations is in good agreement with that previously determined from histological studies.2. The myofibrils are composed of bundles of myosin filaments ranging in width from about 50 to 250 A and extending continuously and in relatively straight lines through the isotropic and anisotropic bands in both the extended and contracted states. The anisotropic bands also contain material of high electron scattering power and affinity for phosphotungstic acid. The distribution of this "A substance" changes with contraction in characteristic fashion. The evidence indicates that the myosin filaments are the contractile units.3. While the myosin filaments have an indefinite length in the intact fibril, they are fragmented extensively during extraction in weakly alkaline salt solutions (method of Greenstein and Edsall). Filam...

An investigation of cross striations and myosin filaments in muscle.

The theoretical limit of resolving power for transmission electron microscopes is in the range 5 to 10 A and it is not unusual for resolutions in the range 10 to 20 A to be reported in practice. This means that the dimensions of a large dass of biologically important molecules and synthetic polymers should be directly observable if contrast and other limitations could be overcome. For example, a spherical protein molecule with a diameter of 20 A would have a molecular weight of only about 3500. Except for a few isolated instances, however, electron microscopy as a means for determining molecular size and shape has largely been confined to the study of very large macromolecules which have molecular weights of several millions and dimensions of several hundred angstroms. The reasons for the relative ineffectiveness of the electron microscope for the study of the smaller organic particles are their low electron-scattering power, the difficulty of distinguishing them from their surroundings or substrate, and in many instances the destruction or distortion of structure resulting from the removal of water.

/pdf/method-for-the-observation-of-macromolecules-with-the-5ejdq3bx8j.pdf

Method for the Observation of Macromolecules with the Electron Microscope Illustrated with Micrographs of DNA

The visualization of polyribosomal structure.

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