Search PubMed⌕ Search

Biomedical subjects

D D Shukla

Publications and source records attributed to D D Shukla.

42 records · Page 3Linked to original sources
PubMed1983

Purification of barley yellow dwarf virus by gel filtration on Sephacryl S-1000 Superfine.

Barley yellow dwarf virus was purified from infected oats using cellulase to assist virus extraction, clarification of the extract with chloroform-butanol, precipitation of virus by polyethylene glycol and gel filtration of the resulting suspension on Sephacryl S-1000 Superfine. The virus was further purified by sucrose density gradient centrifugation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electron microscopy showed that the virus preparation is of sufficient purity for biochemical investigation. A yield of approximately 0.8 mg virus/kg of infected leaf was obtained. The technique is simple and less time-consuming than conventional centrifugation procedures and can be used routinely for purification of a wide range of spherical plant viruses.

Acrylic Resins↗
PubMed1982

Electro-blot radioimmunoassay of virus-infected plant sap - a powerful new technique for detecting plant viruses.

A new technique for detecting viruses in plant sap is described. It consists of sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the infected plant sap, electrophoretic transfer of protein bands to activated paper by the Electro-Blot technique, the subsequent probing of the viral coat protein band by specific antiserum (prepared against intact virus), and detection of immune complex with 125 I-labelled protein A. The technique successfully detected tobacco mosaic virus at a sap dilution of 1 : 10,000, four strains of sugarcane mosaic virus (a potyvirus) in their perennial hosts infected for about 4 years, and five different isolated of potato leaf roll virus (a luteovirus). The latter virus occurs in extremely low concentration and is difficult to detect by the other known methods.

Electrophoresis, Polyacrylamide Gel↗
PubMed1980

Further studies on the use of protein A in immune electron microscopy for detecting virus particles.

The immune electron microscopic technique which involves pre-coating electron microscope grids with protein A before coating them with the specific antiserum, has been found suitable for detecting isometric insect and plant viruses. With the three virus-antibody combinations tested, the optimum antiserum dilution for protein A plus antiserum treatment was found to be 1:100 or less, whereas in the case of grids treated with antiserum alone it ranged from 1:1000 to 1:2000 although the titre of the antisera ranged from 1:512 to 1:4096. The increase in the number of particles on grids treated with protein A plus antiserum over those treated with antiserum alone, at each optimal antiserum dilution, was 25.7-fold (sugarcane mosaic virus), 2.1-fold (tobacco mosaic virus) and 1.6- and 2.4-fold (Erysimum latent virus--two different sap dilutions). Protein A plus antiserum-coated grids can be stored for up to 6 months at 4 degrees C, but not at room temperature or in a desiccator, and still retain about 25% of their activity, sufficient to detect any virus using the electron microscope. Antisera preserved in glycerol can be used successfully for detecting viruses by immune electron microscopy.

Immunologic Techniques↗
PubMedDate not supplied

Purification of hydrophilic and hydrophobic peptide fragments on a single reversed phase high performance liquid chromatographic column.

Hydrophilic peptides generated from enzymic fragmentation of proteins are difficult to purify because they are either weakly bound or unretained by the reversed phase C18 columns favoured for liquid chromatographic separation of peptide mixtures. To overcome this difficulty, peptides that were not bound or only weakly bound by a C18 RP column were reacted with phenyl isothiocyanate (PITC), as used in the initial step in Edman sequencing. The hydrophobic phenylthiocarbamyl (PTC) peptide derivatives produced by the reaction were rechromatographed on the same column. Peptides generated by tryptic digestion of equine cytochrome C were used as a model system to test whether a complete set of peptide fragments could be purified by this method using just one column and solvent system. All the expected hydrophobic tryptic peptides bound to the RP column and were resolved by elution with acetonitrile, but no hydrophilic peptides were recovered as pure fractions. The column breakthrough fraction was reacted with PITC and rechromatographed on the same column, producing a profile consisting of 19 bound peaks. Further rechromatography of some of the fractions at different column temperatures enabled all six of the expected hydrophilic peptides to be purified and identified. The technique has also been applied to the sequence determination of coat protein from peanut stripe potyvirus protein, eight hydrophilic tryptic peptides being recovered and identified as PTC derivatives.

Amino Acid Sequence↗