A rapid method for the purification of antibody--enzyme conjugates.

A method is described for a rapid and efficient separation of enzyme-labelled antibodies from the free enzyme following the coupling reaction. A single passage of the reaction mixture on a protein A--Sepharose CL-4B column gave a sharp separation of the free enzyme from the conjugate.     

A rapid purification method for human erythrocyte pyruvate kinase

Human erythrocyte pyruvate kinase (ATP: pyruvate phosphotransferase, E.C.2.7.1.40) is purified 30,000-fold, using a method which includes ammonium sulfate precipitation, Sephadex G-75 filtration, and Blue Dextran-Sepharose 4B chromatography. The enzyme is resolved into two peaks on Blue Dextran-Sepharose 4B. The first peak with sp act of 300 corresponds to the mature form (R4) whereas the second peak with sp act of 180 corresponds to R2R'2. Peaks I and II give one band on 10% polyacrylamide gel without SDS. Peak II gives two bands on 10% SDS gel with molecular weights 60,000 (R') and 57,500 (R). On the other hand, peak I gives only one band on 10% SDS gel having a molecular weight of 57,500. Both the R4 and R2R'2 forms of the enzyme have the same pH optimum of 7.2.     

A rapid method for extraction and purification of DNA from dental plaque.

A rapid method based on previously described DNA extraction procedures was developed for the isolation of DNA from dental plaque samples. The isolated DNA is suitable for use in the PCR. Freeze-thawing, cell wall-degrading enzymes, and guanidine isothiocyanate were used to lyse cells and release DNA. The released DNA was adsorbed onto diatomaceous earth and purified by washing with guanidine isothiocyanate, ethanol, and acetone. The purified DNA was released from the diatomaceous earth into an aqueous buffer and analyzed by PCR with 16S rDNA primers (rDNA is DNA coding for rRNA). As judged from studies with pure cultures of a number of bacterial species, gram-negative and gram-positive organisms were lysed equally well by this procedure. The amount of PCR product was proportional to the number of cells analyzed over the range tested, 500 to 50,000 cells. On the basis of studies with plaque samples that were spiked with known quantities of the oral bacterium Treponema denticola, the DNA prepared from plaque was free of substances inhibitory to PCR. This method should have utility in molecular genetic studies of bacterial populations not only in uncultured plaque samples but also in other complex bacterial assemblages.     

A rapid method for the purification of extrachromosomal DNA from eukaryotic cells.

A simple and efficient procedure to purify the low molecular weight extrachromosomal DNA from eukaryotic cells is described. Gentle lysis of cells with urea and sodium dodecyl sulfate in 0.24 M phosphate buffer (pH 6.8) is followed by the removal of high molecular weight bulk DNA by centrifugation. Protein and RNA are removed from the supernatant by hydroxyapatite chromatography in urea/phosphate buffer. Urea is then removed with 0.15 M phosphate buffer and the extrachromosomal DNA, virtually free from protein and RNA, is finally eluted in 0.5 M phosphate buffer. The procedure allows the recovery of about 99% simian virus 40 (SV40) DNA from infected monkey kidney cells in the extrachromosomal fraction. In normal mouse, monkey, andhuman cells, approximately 1% of total cell DNA appears to be extrachromosomal.     

A rapid and efficient method for the purification of tyrosinase from Neurospora.

A short and efficient procedure consisting of two chromatographic steps is described for the isolation of tyrosinase from Neurospora. The first step, Celite-column chromatography, resulted in the isolation of four copper-containing proteins from the crude mycelial extract. Anion-exchange chromatography on DEAE-Sephadex of these proteins resulted in the isolation of electrophoretically and serologically pure tyrosinase. More than 70% of the initial tyrosinase activity was recovered in the final enzyme preparation, which had a specific activity of 450 units/mg.     

A rapid method for purification of arylsulphatase A from rabbit uterus

The arylsulphatase A from rabbit uterus has been purified with a rapid method using Fast Protein Liquid Chromatography (FPLC). The enzyme is an acid glycoprotein with an apparent molecular weight of 110,000. The enzymatic activity is competitively inhibited by various phosphoesters and various ions such as SO4(2-), PO4(3-) and P2O7(4-).     

A rapid purification method of restriction endonucleases from Haemophilus strains.

A simple and rapid method of purification of restriction endonucleases from different Haemophilus strains is presented. By this method highly purified and stable enzymes can be obtained. Separation of different restriction activities present in the same strain is possible. This method was so far successfully used with Haemophilus influenzae, Haemophilus parainfluenzae and Haemophilus aegyptius strains. The main advantages over previously published procedures reside in the simplication of certain purification steps (for instance the BioGel A 0.5 M filtration is replaced by a hydroxyapatite batch step), elimination of exonuclease activity by fractionation with (NH4) 2SO4, separation of different restriction activities by phosphocellulose chromatography, application of this method to various strains and high purification degree of enzymes.     

A simple and rapid purification method for Escherichia coli DNA polymerase I.

We report a simple, three-step method for the purification of Escherichia coli DNA polymerase I. Its advantages over other procedures are ease and rapidity, the absence of an autolysis or any high speed centrifugation step, and applicability to large quantities of material. In addition, RNA polymerase can be isolated as a by-product. We have applied this method to purify DNA polymerase both from wild type E. coli cells and from cells bearing a lambda prophage carrying the polA gene (Kelley, W.S., Chalmers, K., and Murray, N.E. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5632-5636). This latter source amplifies the amount of DNA polymerase in the cells by at least 10-fold.     

A rapid, cost-effective method of assembly and purification of synthetic DNA probes >100 bp

Here we introduce a rapid, cost-effective method of generating molecular DNA probes in just under 15 minutes without the need for expensive, time-consuming gel-extraction steps. As an example, we enzymatically concatenated six variable strands (50 bp) with a common strand sequence (51 bp) in a single pool using Fast-Link DNA ligase to produce 101 bp targets (10 min). Unincorporated species were then filtered out by passing the crude reaction through a size-exclusion column (<5 min). We then compared full-length product yield of crude and purified samples using HPLC analysis; the results of which clearly show our method yields three-quarters that of the crude sample (50% higher than by gel-extraction). And while we substantially reduced the amount of unligated product with our filtration process, higher purity and yield, with an increase in number of stands per reaction (>12) could be achieved with further optimization. Moreover, for large-scale assays, we envision this method to be fully automated with the use of robotics such as the Biomek FX; here, potentially thousands of samples could be pooled, ligated and purified in either a 96, 384 or 1536-well platform in just minutes.     

A rapid method for analyzing the ligation products of synthetic oligodeoxyribonucleotides

A method is described for the rapid analysis of DNA ligation products in the assembly of synthetic genes and gene fragments. The method is based on the simultaneous analysis of multiple ligation reactions where a single but different DNA oligomer is radiolabelled per ligation reaction. After each ligation the reaction mixture is electrophoresed on a denaturing, as well as a non-denaturing, polyacrylamide gel allowing one to monitor the ligation reaction products. In addition, a unique method for generating single stranded DNA sizing standards up to approximately 300 nucleotides in length is described.     

A rapid procedure for purification of EcoRI endonuclease.

A convenient and rapid procedure has been developed to purify restriction endonuclease Eco RI. The method involves sonication of cells at low ionic strength, precipitation of the endonuclease with Polymin P (a polyethyleneimine), elution of the enzyme from the Polymin P precipitate, ammonium sulfate precipitation and chromatography on phosphocellulose. The purified restriction endonuclease is free of exonuclease and other endonucleases.     

A rapid and convenient method for purification and isolation of chlorophyll-A from Porphyra yezoensis.

A rapid and convenient method for purification and isolation of chlorophyll-a from Porphyra yezoensis without any chromatographic procedures has been developed. Thin-layer chromatographic and high-performance liquid chromatographic tests revealed that chlorophyll-a preparations in this study did not contain any other photosynthetic pigments and their degradation products.     

A rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens.

This paper describes a simple and rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens, together with analysis of the kinetic behaviour and some properties of the enzyme. The purification consisted of two steps, 2',5'-ADP-Sepharose 4B affinity chromatography and DEAE Sepharose Fast Flow ion exchange chromatography in procedure which took two working days. The enzyme was obtained with a yield of 13.7% and had a specific activity of 2.64 U/mg protein. The overall purification was about 19,700-fold. The molecular weight of the enzyme was found to be 62 +/- 3 kDa by Sephadex G-200 gel filtration chromatography. A protein band corresponding to a molecular weight of 69.2 +/- 3.2 kDa was obtained on SDS polyacrylamide slab gel electrophoresis. On chromatofocusing, lens glucose-6-phosphate dehydrogenase gave a single peak at pI 5.14. The activation energy of the reaction catalyzed by the enzyme was calculated from Arrhenius plot as Ea = 5.88 kcal/mol. The pH versus velocity curve had two peaks at pH 7.7 and 9.6. By the double-reciprocal plots and the product inhibition studies, it was shown that the enzyme follows 'Ordered Bi Bi' sequential kinetics. From the graphical and statistical analyses, KmNADP+, KmG-6-P, KiNADPH, Ki6-PGA were estimated to be 0.008 +/- 0.002, 0.035 +/- 0.013, 0.173 +/- 0.007 and 1.771 +/- 0.160 mM, respectively. The observed kinetic behaviour of glucose-6-phosphate dehydrogenase from bovine lens was in accordance with the enzyme from other sources.