Ribosome staining

The life span of the normal red blood cell is 120 days this means that slightly less than 1% of the population of red cells (200 billion cells, or 2 million per second) is replaced daily. The new red cells that appear in the circulation still contain ribosomes and elements of the endoplasmic reticulum. The RNA of the ribosomes can be detected by suitable stains (such as cresyl blue), and cells containing it are termed reticulocytes they normally number about 1% of the total red blood cell count. The life span of the red blood cell can be dramatically shortened in a variety of hemolytic anemias. The number of reticulocytes is markedly increased in these conditions, as the bone marrow attempts to compensate for rapid breakdown of red blood cells by increasing the amount of new, young red cells in the circulation. 

E. F. DeLong, G. S. Wickham, and N. R. Pace, Phylogenetic stains ribosomal RNA-based probes for the identification of single cells. Science 245 1360 (1989). 

Lead citrate/uranyl acetate6 Step 1 Float or immerse sections for 10-30 min on filtered 1-2% aqueous uranyl acetate (or in EtOH) wash with ultrapure H20 (three beakers of 50 mL each) by dipping grids held with a forceps dry for 5 min Step 2 Place drops of lead citrate (lead carbonate free) onto a wax surface (parafilm or dental wax) in a Petri dish line edges of dish with pellets of KOH float grid with sections (sections face down) for 4-5 min (if overstained 2-3 min and dilute stain) wash grids with sections in ultrapure H20 Nonselective enhancement of membrane contrast, ribosomes, and nuclear material proteins and lipid droplets... 

Khulbrandt W, Unwin PNT. Structural analysis of stained and unstained two-dimensional ribosome crystals, in Electron Microscopy at Molecular Dimensions, State of the Art and Strategies for the Future (Baumeister W, Vogell W, eds.), Springer-Verlag, Berlin, Germany, 1980, pp. 108-116. 

Figure 7.7. Agarose gel electrophoresis of total RNA. Total RNA from mouse skin (panel a, lane 2) and two human cadaver skin samples (panel b, lanes 1 and 2) were isolated by guanidine thiocyanate method and size fractionated on denaturing formaldehyde containing 1% agarose gel and stained with 0.5 pg/mL ethidium bromide. Note that in case of mouse skin RNA, two distinct ribosomal RNA bands (upper 28S and lower 18S bands) are clearly visible. In contrast, in case of human skin samples, which were collected several hours postmortem, there is partial RNA degradation as is evident by fuzzy 28S and 18S ribosomal RNA bands. RNA degradation is more pronounced in one of the samples than the other (panel b, compare lane 1 and lane 2). Ribosomal RNA bands are indicated by arrowheads. RNA size markers (Invitrogen, Carlsbad, CA) in the range 0.24 to 9.5 kb are in lane 1 (panel a) and lane 3 (panel b).

Studies of KH clearly indicate complexity that is only partially resolved (49). DiflFerential staining reveals small, dense, homogeneous particles within amorphous KH masses, usually associated with tono-fibrils (32, 48, 49). Amino acid analyses of supposed KH materials show at least three distinctive patterns (see Table I). The amorphous material of Tezuka and Freedberg (72) has much less proline and cystine than KH studied by Matoltsy (51). Other workers have associated histidine with KH in granular cells (48, 49, 76). Tezuka s histidine values (72) fall between those of Matoltsy (51) and Hoober (76) and conceivably represent an analysis of mixed components. UgeFs bovine material is a nucleoprotein that may be either a ribosomal product or still another KH component (71, 77). 

The most characteristic feature of a eukaryotic cell, the nucleus, consists of a nucleoplasm surrounded by a double nuclear m mbrane pierced by nuclear pores. The nucleoplasm contains the (linear) chromosomes of the cell, which are organised into heterochromatin, which stains only a little, and euchromatin, which stains more densely. The most important euchromatic area is the nucleolus, in which ribosomes are formed (Fig. 1.2). 

The ability of gel electrophoresis to discriminate between conformational states that may differ in only minor degree in Stokes radius can be put to a number of good uses, for example in measuring the equilibria between the conformers of tRNA. Richards et al. (1973), working with 5 S RNA, have used gel electrophoresis to study the kinetics of interconversion between the native state (defined by its ability to recombine with the 50 S ribosome) and the denatured. Although these forms are practically identical in the extent of base-pairing, they separate readily in gel electrophoresis, and their relative proportions can be measured by densitometry of the stained... 

Fig. 10.16. Detection of binding of a ribosomal protein to 16 S ribosomal RNA. The presence of protein S 4 in the 16 S zone, shown in electrophoresis result (A), causes it to give a positive response to the stain Coomassie Brilliant Blue represented photographically in B and densitometrically in C (Garret et al. 1971).

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