Guanidine thiocyanate

Chemical Properties. Ammonium thiocyanate rearranges upon heating to an equiHbrium mixture with thiourea 30.3 wt % thiourea at 150°C, 25.3 wt % thiourea at 180°C (373,375). At 190—200°C, dry ammonium thiocyanate decomposes to hydrogen sulfide, ammonia, and carbon disulfide, leaving guanidine thiocyanate [56960-89-5] as a residue. Aqueous solutions of ammonium thiocyanate are weakly acidic a 5 wt % solution has a pH of 4—6. 

After 2 h incubation of the prepared antibody beads with UV-crosslinked extract in a cold room, the beads are washed 4 x with 100 /A RIPA buffer (50 mMTris-HCl pH 7.5, 150 rnMNaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) and lx with genomic DNA lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 2.5 mM DTT, 0.5 mM spermidine, 1% Triton X-100). Approximately 300 /(I of PK solution (1 mg/ml proteinase K in genomic DNA lysis buffer and 0.2 U//A RNase inhibitor) is added to the total lysate previously kept on ice and the beads are then incubated at 37° for 30 min. Gently flick the tubes to resuspend the beads every 10 min during the incubation. After removal of the proteinase K solution, 300 /A of RNA extraction solution (4 M guanidine thiocyanate, 0.5% sarkosyl, and 25 mM sodium citrate, pH7) is added to the beads, incubated for 10 min and the supernatant is mixed with 30 fig yeast tRNA (as a carrier) and 30 fil of 3 M sodium acetate. The RNA solution is phenol-chloroform extracted, ethanol-precipitated, and the pellet washed once with 70% ethanol. The dry pellet is used for 1st strand cDNA synthesis, followed by PCR analysis. The removal of proteins... 

Other major reagents TRIzol LS reagent (Invitrogen Co., Carlsbad, CA), a commonly used commercial reagent for RNA extraction from cell/tissuc sample that contains phenol and guanidine thiocyanate 0.1 M sodium hydroxide (NaOH) solution is used for DNA extraction. 

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. 

The lysis solution contains guanidine thiocyanate (GuSCN), which is an irritant to the respiratory tract and skin. Experimental steps involving this solution should therefore be performed under a hood, and gloves should be worn. 

The equihbriuin mixtures at 150°C and 180°C contain 30.3% and 25.3% (by weight) thiourea, respectively. When heated at 200°C, the dry powder decomposes to ammonia, hydrogen sulfide, and carbon difsuUide, leaving a residue of guanidine thiocyanate [56960-89-5]. 

Like the inorg compds of the azido acid, and unlike the alkyl and aryl derivs, the guanidine salt is photosensitive. In the dark it may be stored below 10°, in vacuo, for days without appreciable decompn. On long standing at RT, the salt decomps quanty, yielding Na, sulfur and guanidine thiocyanate. In aq soln the azido salt reacts with AgNOs to form the insol Ag azidodithiocarbonate... 

At the same time some guanidine thiocyanate, NH C(NH2)2.HSCN, is produced, although a larger proportion of this substance is formed at 170° to 180° C.4 The thiourea is freed from guanidine thiocyanate and unchanged ammonium thiocyanate by treatment with cold water, in which thiourea is but sparingly soluble it may then be purified by recrystallisation from water. 

Nitroguanidine may be prepared by the nitration of guanidine salts by means of nitric acid,1 or by the action of concentrated sulfuric acid upon guanidine nitrate.2 The nitration of guanidine thiocyanate yields a product which retains a small proportion of sulfur compounds, and Lhe nitration of the sulfate requires vigorous treatment and gives poor yields. The present procedure, which yields the alpha form,3 is simple and economical, and furnishes a good yield. 

For many years guanidine thiocyanate was the most easily prepared and the most commonly used of the salts of guanidine. Other salts were made from it by metathetical reactions. Nitro-guanidine, prepared from the thiocyanate by direct nitration with mixed acids, was found to contain traces of sulfur compounds which attacked nitrocellulose and affected the stability of smokeless powder, and this is one of the reasons why nitroguanidine powders did not come into early use. Guanidine thiocyanate is deliquescent. Strong solutions of it dissolve filter paper. 

Colorless guanylthiourea melts to a pale yellow liquid, which resolidifies to a yellow crystalline mass on cooling partial rearrangement to guanidine thiocyanate occurs during the fusion. 

PICRIC ACID-STEAM AUTOCLAVING-FORMIC ACID-GUANIDINE THIOCYANATE-ASSISTED RETRIEVAL OF PRION PROTEIN... 

Great care should be taken in handling the tissue to avoid infection. Picric add is both toxic and explosive. Safety guidelines must be used when working with this reagent. Guanidine thiocyanate is also a biohazardous material. 

Fusion of ammonium thiocyanate,6,6 either alone or in the presence of heavy metal salts, at 180°C., gives guanidine thiocyanate. This procedure has been modified by heating lead nitrate with a solution of ammonium thiocyanate in liquid ammonia at 120°.7 Lead sulfide is formed, and guanidine nitrate recovered. Other methods of formation involve the hydrolysis of dicyandiamide8-10 and the nitridation11 of methyl amine by means of ammonium azide in liquid ammonia. 

Although guanidine thiocyanate is readily prepared by this method, it is extremely deliquescent and cannot be obtained in so pure a state as guanidine nitrate. 

Liquid-liquid extraction of polynucleotides normally involves the use of an organic solvent (such as phenol or chloroform) or an aqueous solution (such as sodium perchlorate, guanidine hydrochloride or guanidine thiocyanate in high concentration) to remove the proteinaceous materials from the aqueous phase... 

St.Louis, MO), and RNA STAT-60 (TEL-TEST B, Inc, Friendswood, TX), or similar ready-to-use RNA extraction reagents. It is also possible to isolate RNA, DNA, and proteins simultaneously from the tissue homogenate using guanidine-thiocyanate and phenol [20,21], Since DNA is trapped in the interphase and the phenol phase under acidic pH during extraction, the addition of an equal volume of 1 M Tris solution (pH 10.5) to the phenol phase raises the pH and thereby increases the solubility of the DNA in the aqueous phase. This DNA can be recovered by ethanol precipitation. 

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).

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