Contamination with proteins

The upper aqueous phase containing nucleic acids is then separated and the DNA precipitated by addition of ethanol. Because of the ionic nature of DNA, it becomes insoluble if the aqueous medium is made less polar by addition of an organic solvent. The DNA forms a threadlike precipitate that can be collected by spooling onto a glass rod. The isolated DNA may still be contaminated with protein and RNA. Protein can be removed by dissolving the spooled DNA in saline medium and repeating the chloroform-isoamyl alcohol treatment until no more denatured protein collects at the interface. 

As the extracted LPS is always contaminated with proteins, specially lipoproteins, additional hydrolysis by proteinase and other purification processes have been... 

In general, DNA contamination with protein can be calculated using the following equation ... 

Determine the plasmid yield spectrophotometrically. Dilute an aliquot of the DNA in water and measure the optical density (OD) at 260 nm, which allows the calculation of the concentration of nucleic acid in the sample. An OD of 1 at 260 nm corresponds to approx 50 pg/mL for double-stranded DNA. The ratio between the readings at 260 and 280 nm (OD26o/OD28o) provides an estimate of the purity of the nucleic acid. Pure preparations of DNA have ratios of 1.8. If there is contamination with protein, the OD260/OD2Xll will be significantly less than 1.8, and accurate quantification of the amount of DNA will not be possible. 

The peak fractions of protein eluting at 0.2 M NaCl are individually evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (see Note 10). Pooled fractions should be >90% pure and not be contaminated with proteins of similar molecular weight. Otherwise, the contaminants will still be present after sizing chromatography. 

We have found it essential to use highly purified DNAs for quantitative hybridization. In particular the labelled reference DNA must be free from contaminating cellular material. Conventional phenol-chloroform, RNAse treatments yield DNA which is free from contamination with proteins and phenol and has good spectrophotometric ratios (258 230 and 258 280nm) but it may contain as much polysaccharide as it does DNA ... 

The absorbance of the RNA at 260 nm and 280 nm should be measured in quartz cuvets. The 260/280 ratio should be equal or close to 2.0. Significant contamination with protein will result in 260/280 ratio lower than this. If the ratio is below 1.9, extract the samples with equilibrated phenol and recover by ethanol precipitation as in 6-8. An A260 of 1 is equivalent to a RNA concentration to a 37 pg/mL. Store the RNA solution at -70°C or ethanol precipitate aliquots at -70 C to be recovered as desired (see Note 5). 

Once resuspended, take a small volume of the sample, dilute it in a volume of water according to the size of the spectrophotometer quartz cuvet available, and measure the absorbance at 260 nm. Consider that 1 OD = 40 mg/mL of RNA. Absorbance at 280 nm should indicate contamination with proteins and/or phenol. A good-quality RNA should have a ratio of = 1.8-2.0. 

Gum arable is always contaminated with proteineous matter. Hydroxy-proline, serine, and proline are the most abundant amino acids in the protein-... 

In wool scouring, the contaminants on the wool, mainly grease, dirt, suint, and protein material, are washed off the fiber and remain in the wastewaters either in emulsions or suspension (grease, dirt, protein) or in solution (suint). Centrifugal extraction of the wastewaters produces a grease contaminated with detergent and suint. This product is called wool grease. 

Microbial growth incomplete regeneration contamination with (Lipo)proteins or dyes... 

Columns can be washed with solvents and solvent combinations suitable to remove adsorbed contaminants. When considering the adsorption of analytes, think not only of the diol functionality, but also of the adsorption to residual silanols. Often, the injection of small amounts (500 /d) of dimethyl sulfoxide removes contamination that has accumulated on the column. Aqueous solutions of sodium dodecyl sulfate, guanidine hydrochloride, or urea are compatible with Protein-Pak columns. 

In contrast to these results, Ross et al. [33] found that antisera against a 20-amino acid peptide (Ser-613-Arg-632) of the cytoplasmic domain of the human Na /H exchanger recognized a 66-kDa protein in immunoblots of bovine renal brush border membranes. Since the purity of these membranes was not reported it is possible that this result was due to contamination with basolateral membranes (although the molecular mass would still differ from the basolateral Na /H exchanger in LLC-... 

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