Effects of dextrans on immunoprecipitation in agar and in low-temperature-gelling agarose gels

Hyslop, N. St. G.; Cochrane, Delma

doi:10.1016/0022-1759(74)90094-5

Cited by 3 publications

(3 citation statements)

References 11 publications

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“…Triton X-100 was used in this system to enable better comparison of envelope and cytoplasmic antigens in terms of relative mobility, especially with the use of intermediate gels. Its inclusion also permitted considerable saving (up to 25%) in the volumes of antisera that had to be added to gels to get all immunoprecipitates within scale due to depression of peak heights and enhancement of immunoprecipitation, perhaps in a somewhat analogous manner to the enhancement effected by dextrans and polyethylene glycol (26,53).…”

Section: Resultsmentioning

confidence: 99%

Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis

Smyth¹,

Friedman‐Kien²,

Saltón³

1976

Infect Immun

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Crossed immunoelectrophoresis was used to study two complex antigenic preparations from Neisseria gonorrhoeae, one of cytoplasmic origin and the other derived by Triton X-100 extraction of isolated washed gonococcal envelopes, with the aim of developing suitable reference antigen-antibody systems that could be subsequently used to investigate the immune response to gonococcal infection and to monitor envelope preparations for cytoplasmic contamination. A number of parameters were investigated to optimize and standardize antigen preparation, e.g., harvesting and washing of gonococci, methods of bacterial disruption, and washing of envelopes. The effects of Triton X-100 concentration, initial total envelope protein concentration, and the composition, pH, and concentration of buffer on cell envelope extractability were studied to obviate the need to concentrate material before use in crossed immunoelectrophoresis. The electroendoosmotic properties of agarose were a major determining factor in resolving envelope antigens. From 25 to 30 immunoprecipitates were revealed in the envelope antigen-antibody system; 75 to 80 were revealed in the cytoplasmic system. Envelope immunoprecipitates with reduced nicotinamide adenine dinucleotide and lactate dehydrogenase activities were identified. Crossed immunoelectrophoresis with intermediate gels revealed the presence of antibodies in a preimmune rabbit antiserum pool to a distinctive fast-moving component in both the envelope and cytoplasmic antigen preparations. The intermediate gel technique also demonstrated that extensive washing of envelope preparations with buffer did not remove cytoplasmic contamination completely. The method provides a much more sensitive means of monitoring the purity of envelope fractions than the use of single enzyme markers as indexes of such contamination. The use of rabbit antisera raised to formolized gonococci in intermediate gels indicated that both reference antigen-antibody systems were of potential use in screening immune responses to N. gonorrhoeae. 1273 on July 16, 2020 by guest http://iai.asm.org/ Downloaded from 1274 SMYTH, FRIEDMAN-KIEN, AND SALTON

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Section: Resultsmentioning

confidence: 99%

Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis

Smyth¹,

Friedman‐Kien²,

Saltón³

1976

Infect Immun

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“…Agarose, which is a neutral constituent of agar, has a low gelling temperature and is transparent -both qualities essential for an embedding medium. Because of these, and other qualities, such as low fluorescent background and adequate gel strength, agarose has been successfully used in gel electrophoresis (Elevitch et al, 1966), radial immunodiffusion (Werner, 1967), gel filtration (Werner, 1966), and immunoprecipitation tests (Hyslop and Cochrane, 1974). Here, we demonstrated that agarose can also be successfully used for embedding single cells prior to postfixation in O,O, and further processing and embedding in Epon.…”

Section: Advantages O F the Proceduresmentioning

confidence: 55%

An improved method for processing single cells for electron microscopy utilizing agarose

Yuan

Gulyas

1981

Anat. Rec.

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An improved method is presented for processing single cells for electron microscopy. Agarose, which has a low (30 degrees C) gelling temperature, was used as an initial embedding medium for single cells (spermatozoa and oocytes) and dissociated cell preparations (luteal cells and spleen cells). Dispersed cells of corpus luteum, spleen, and epididymal spermatozoa were placed in 1.5% agarose after aldehyde fixation. These fixed cells, embedded in agarose, were packed into a dense pellet by centrifugation, postfixed, then embedded in Epon. Mammalian eggs were not centrifuged; instead, they were embedded in agarose discs. Cells embedded in agarose were cooled below 30 degrees C to allow for gelling, then processed for electron microscopy. Because agarose has a low gelling temperature, some heat-labile substances were preserved, as demonstrated by retention of peroxidase activity using the DAB histochemical method. The agarose embedding procedure is both rapid and facile, and has proven to be of value in the handling of fragile single cells for electron microscopic studies.

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“…Antisera from the rabbits were then tested concurrently in agar and agarose gels against the vegetable or meat antigens of each extract, optimal dilutions being used for the homologous reactants. The weaker arcs of the heterologous vegetable antigen-antibody reactions were enhanced by treatment of the gel with 6 % solutions of Dextran (MW 500,000) as described recently (Hyslop & Cochrane, 1974). Precipitation arcs were stained in Ponceau S, the gels were decolorized, and then dried for storage or for photography.…”

Section: Immunodiffusion Testsmentioning

confidence: 99%

Extraction methods and test techniques for detection of vegetable proteins in meat products:I. Qualitative detection of soya derivatives

Ns¹

1976

J. Hyg.

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SUMMARYExtracts of 3 soya bean preparations, used commercially in certain countries to replace part of the meat in popular meat products, were made by treatment with (i) sodium dodecyl sulphate, (ii) Triton-X100 or (iii) n–Butanol. Similar extracts were made from beef and pork.All extracts were examined by electrophoretic and immunological techniques. Stained polyacrylamide gels revealed distinctive protein bands after electrophoresis. The migration rates of corresponding bands differed between beef and pork extracts. However, the migration rates of vegetable bands revealed certain similarities, but differed very greatly from those of animal origin. Characteristic fast-migrating S-bands were distinguishable only in extracts of vegetable protein. Immunodiffusion tests, using antisera produced in rabbits against each extract, revealed varying degrees of similarity between extracts of vegetable origin, but the antisera were specific for either vegetable or animal protein.

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Effects of dextrans on immunoprecipitation in agar and in low-temperature-gelling agarose gels

Cited by 3 publications

References 11 publications

Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis

Antigenic analysis of Neisseria gonorrhoeae by crossed immunoelectrophoresis

An improved method for processing single cells for electron microscopy utilizing agarose

Extraction methods and test techniques for detection of vegetable proteins in meat products:I. Qualitative detection of soya derivatives

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