Differential Precipitation of Proteins

2019 ◽  
pp. 115-208 ◽  
Author(s):  
Fred Rothstein
Keyword(s):  
Differential Precipitation

An improved radioimmunoassay of C-peptide and its application in a multiyear study.

1989 ◽  
Vol 35 (1) ◽  
pp. 37-42 ◽  
Author(s):  
J E Myrick ◽  
E W Gunter ◽  
V L Maggio ◽  
D T Miller ◽  
W H Hannon
Keyword(s):  
Nonspecific Binding ◽  
C Peptide ◽  
Freeze Thaw ◽  
Serum Pool ◽  
Hands On ◽  
Antibody Complex ◽  
Analytical Range ◽  
Antigen Antibody ◽  
Free Serum ◽  
Differential Precipitation

Abstract A commercial radioimmunoassay (RIA) for human proinsulin C-peptide was modified to improve its ruggedness and specificity, to decrease the influence of specimen matrix, and to shorten "hands-on" time. In the new protocol, we prepare calibrators in a C-peptide-free serum pool, prepared by treatment with activated charcoal (biological matrix), instead of in a defined matrix. This yielded essentially 100% analytical recoveries for C-peptide concentrations up to 300 pmol/L, a broader analytical range. We also corrected calibrators and unknown samples for nonspecific binding (NSB). Decreasing the concentration of ethanol (from 950 to 880 mL/L) for differential precipitation of the antigen-antibody complex resulted in an NSB of less than 10%, while maintaining high bound/total count percentages for samples and calibrators. C-peptide is thermally unstable without aprotinin at -20 degrees C and with or without aprotinin at 4 degrees C or above, but multiple freeze-thaw cycles do not affect C-peptide in serum. The modified C-peptide assay was applied to plasma from a multiyear study (fasting and post-carbohydrate-challenge subjects). During the four years of the study CVs ranged from 1.9% to 8.6% for replicate analyses of C-peptide in samples with concentrations less than or equal to 500 pmol/L. Between-run CVs were 3.8% to 8.2%, total CVs 3.8% to 10.7%.

An Apparent Effect Of Size Distribution Of FVIIIR: Ag Multimers On Immunoradiometric Assays

1981 ◽  
Author(s):  
M A Lamb ◽  
H M Reisner ◽  
H A Cooper ◽  
R H Wagner
Keyword(s):  
Complex Formation ◽  
Immune Complex ◽  
Dose Response ◽  
Response Curves ◽  
Crossed Immunoelectrophoresis ◽  
Normal Human ◽  
Dose Response Curves ◽  
Antigen Antibody ◽  
Differential Precipitation ◽  
Immune Complex Formation

Immunoradiometric assays (IRMA) of FVIIIR: Ag from normal and certain variant VWD plasmas have suggested possible antigenic differences in the molecules. Studies reported thus far have used antibody specific for normal FVIIIR: Ag. We have further studied this question of antigenic differences using 2 populations of antibody isolated from an antisera prepared against highly purified human FVIII. Isolated IgG was labeled with [125-I]. The population of labeled IgG “specific” for variant FVIIIR: Ag was separated by immune complex formation with a VWD plasma previously shown, by 2% agarose crossed immunoelectrophoresis, to contain only the lower molecular weight multimers of FVIIIR: Ag. The “specific” labeled IgG was obtained by low pH dissociation and subsequent G-200 chromatography. [125-I] IgG “specific” for normal FVIIIR: Ag was similarly obtained after immune complex formation with pooled normal human plasma. Liquid phase IRMAs were performed using differential precipitation with ammonium sulfate or PEG to separate antigen-antibody complexes from free antibody. Using antibody “specific” for normal FVIIIR: Ag, a lack of parallelism was noted in the dose-response curves of variant plasmas as well as a decrease in maximum antibody bound, as compared to normal. Interestingly, when this antibody was absorbed with the variant VWD plasma and the remaining antibody used in IRMAs, none was bound by either variant or normal plasma.Using antibody “specific” for variant FVIIIR: Ag, a similar lack of parallelism in dose-response curves and a decrease in maximum antibody bound were observed. Therefore rather than antigenic differences as previously implied, these results suggest that the discrepancies noted in IRMAs of variant and normal plasmas are a function of the size of the FVIIIR: Ag multimers.

scholarly journals TRANSCRIPTION ANALYSIS OF GENE EXPRESSION ISLAND VcB V. CHOLERAE BY METHOD OF FULL-GENOMIC SEQUENCING

2018 ◽  
pp. 39-41
Author(s):  
S.O. Vodop'janov ◽  
A.S. Vodop'janov ◽  
R.V. Pisanov ◽  
S.A. Ivanov ◽  
B.N. Mishan'kin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genomic Sequencing ◽  
Lithium Ions ◽  
Transcription Analysis ◽  
Cholera Vibrio ◽  
Total Rna ◽  
Rna Transcripts ◽  
Total Pool ◽  
Differential Precipitation

The aim of the study was to analyze the expression of V. cholerae genes that are part of the VcB island by means of full-genomic sequencing of the transcriptome. The VcB island is localized on the second chromosome in all toxigenic vibrios studied and is absent in the atoxigenic apiliated strains. Two strains of V. cholerae O1 ctxA+ tcpA+ and one strain V. cholerae O1 ctxA– tcpA– were studied. The pool of total RNA vibrios was isolated by a technique based on differential precipitation in the presence of lithium ions. In the total pool of sequenced RNA, RNA encoded in the order of 3 500 by known cholera vibrio genes was identified. In a pool of total RNA from two ctx + tcpA + strains RNA transcripts were found for the five genes included in the VcB island, excluding the VCA0282 gene, previously identified as the ISVch5-transposase. In the the pool of total RNA from the ctx– tcpA– strain no transcripts of the two genes previously described as VCA0282-transposase and VCA0283 were detected. A possible explanation may be the existence of copies of these genes in other parts of the genome of the ctxA– tcpA– V. cholerae.

Differential centrifugation, calcium precipitation, and ultrasonic disruption of midgut cells of Erinnyis ello caterpillars. Purification of cell microvilli and inferences concerning secretory mechanisms

1986 ◽  
Vol 64 (2) ◽  
pp. 490-500 ◽  
Author(s):  
Custódio D. Santos ◽  
Alberto F. Ribeiro ◽  
Walter R. Terra
Keyword(s):  
Subcellular Fractions ◽  
Soluble Form ◽  
Differential Centrifugation ◽  
Posterior Midgut ◽  
Brush Borders ◽  
Columnar Cells ◽  
Differential Precipitation ◽  
Anterior Midgut ◽  
Erinnyis Ello ◽  
Calcium Precipitation

Subcellular fractions of the cells from the first and last third of midguts from Erinnyis ello caterpillars were obtained by conventional homogenization, followed by differential centrifugation or differential calcium precipitation, as well as by partial ultrasonic disruption. Aminopeptidase was enriched in the subcellular fractions, which in the electron microscope display mainly microvilli from the columnar cells (obtained by differential centrifugation and ultrasonic disruption), and also in the microvilli fraction obtained by differential precipitation. To account for the enzyme activities that sedimented with vesicles displaying brush borders, major amounts of the soluble glycosidases (cellobiase, N-acetylglucosaminidase, maltase, and trehalase) are assumed to be loosely bound to the cell glycocalyx, from where they are set free by homogenization and (or) freezing–thawing. Intracellular glycosidases seem to be bounded by membranes, which sediment together with vesicles that resemble secretory vesicles. The soluble form of amylase occurred mainly associated with the microvilli of anterior midgut cells and is supposed to be contained inside small vesicles, which are seen budding along columnar cell microvilli and fusing one with the other and with the tips of the microvilli from the anterior midgut cells. Secretory mechanisms are discussed in the light of the evidence that the posterior midgut secretes whereas the anterior midgut absorbs water.

Fractionation of spectrin by differential precipitation with calcium

1976 ◽  
Vol 175 (1) ◽  
pp. 367-372 ◽  
Author(s):  
F.H. Kirkpatrick ◽  
G.M. Woods ◽  
R.I. Weed ◽  
P.L. La Celle
Keyword(s):  
Differential Precipitation
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