Denaturation guanidine thiocyanate

The cells are lysed in a buffer containing strong chaotropic reagents such as guanidine thiocyanate and 2-mercaptoethanol, which completely denatures any ribonuclease present. The supernatant is then placed on a cushion of CsCl (5.7 mol l-1) and centrifuged at 100000 g for 18 h. The RNA passes through the CsCl and is pelleted, while the DNA and protein remain in the aqueous solution. The RNA pellet is dissolved in buffer and concentrated by precipitation in cold ethanol. 

Although guanidine hydrochloride is a potent chaotropic agent, its use for RNA isolation has not been as popular as that of guanidine thiocyanate. The reasons may be that it needs to be used at a much higher concentration to be an effective protein denaturant. The method described here is a modification of methods described in Refs. 15 and 16. 

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).

AI3-18430 EINECS 209-812-1 Guanidine, monothiocyanate Guanidine thiocyanate Guanidinium thiocyanate Isothiocyanic acid, compd. with guanidine (T.1) NSC 2119 Thiocyanic acid, compd. with guanidine (1 1) USAF EK-705. Potent protein denaturant used in isolation of intact DNA, RNA. Crystals mp= 117°. Dajac Labs. Eastman Chem. Co. Ftuka U.S. BioChem. 

Best dissociation conditions vary with the antibodies (Section 8.2). Most techniques depend on the deformation of the interacting surfaces of the antibody and antigen, obtained with chaotropic ions (3.5 M potassium thiocyanate in 100 mM phosphate buffer, pH 6.6), organic acids with low surface tension (propionic acid, acetic acid. Section 8.2), denaturants (8 M urea, 7 M guanidine-HCl), and pH extremes (100 mM glycine-HCl, pH 2.5). 

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