Human serum protein fractionation by gel filtration.
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ABSTRACT:
SUBMITTER: Freeman T
PROVIDER: S-EPMC1179298 | biostudies-other | 1970 Aug
REPOSITORIES: biostudies-other
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Project description:1. Culture filtrates from Myrothecium verrucaria have been fractionated by gel filtration on Sephadex G-75 to give three major cellulolytic components with molecular weights of about 55000, 30000 and 5300. 2. The middle component has the bulk (90%) of the total carboxymethylcellulase activity and is little affected by exposure to cotton. The other two, which are mainly responsible for the activity of the filtrate towards cotton, are removed or deactivated by exposure to it. These observations accord with the previously reported behaviour of the whole culture filtrate. There is no evidence for interconversion of, or synergism between, these components. 3. Temperature control during gel filtration is necessary for reproducible results at high resolution. The effect of a change in temperature has been explained in terms of changes in the degree of swelling of the gel particles.
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Project description:Mapping Atheroprotective Functions and Related Proteins/Lipoproteins in Size Fractionated Human Plasma
2017-02-23 | PXD005520 | Pride
Project description:The role of sialic acid in the gel-filtration behaviour of sialoglycoproteins was investigated by using the separated isoenzymes of purified human liver alpha-L-fucosidase and several other well-known sialic acid-containing glycoproteins (fetuin, alpha1-acid glycoprotein, thyroglobulin and bovine submaxillary mucin). For each glycoprotein studied, gel filtration of its desialylated derivative gave an apparent molecular weights much less than that expected just from removal of sialic acid. For the lower-molecular-weight glycoproteins (fetuin and alpha1-acid glyocprotein), gel filtration of the sialylated molecules led to apparent molecular weights much larger than the known values. The data indicate that gel filtration cannot be used for accurately determining the molecular weights of at least some sialoglycoproteins.
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Project description:Plasma levels of high density lipoprotein cholesterol (HDL-C) are inversely proportional to the incidence of cardiovascular disease. Recent applications of modern proteomic technologies have identified upward of 50 distinct proteins associated with HDL particles with many of these newly discovered proteins implicating HDL in nonlipid transport processes including complement activation, acute phase response and innate immunity. However, almost all MS-based proteomic studies on HDL to date have utilized density gradient ultracentrifugation techniques for HDL isolation prior to analysis. These involve high shear forces and salt concentrations that can disrupt HDL protein interactions and alter particle function. Here, we used high-resolution size exclusion chromatography to fractionate normal human plasma to 17 phospholipid-containing subfractions. Then, using a phospholipid binding resin, we identified proteins that associate with lipoproteins of various sizes by electrospray ionization mass spectrometry. We identified 14 new phospholipid-associated proteins that migrate with traditionally defined HDL, several of which further support roles for HDL in complement regulation and protease inhibition. The increased fractionation inherent to this method allowed us to visualize HDL protein distribution across particle size with unprecedented resolution. The observed heterogeneity across subfractions suggests the presence of HDL particle subpopulations each with distinct protein components that may prove to impart distinct physiological functions.
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Project description:Mapping Atheroprotective Functions and Related Proteins/Lipoproteins in Size Fractionated Human Plasma
| MSV000081547 | MassIVE
Project description:Fractionation of complex samples at the cellular, subcellular, protein, or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e. 1D SDS-PAGE, 1D preparative SDS-PAGE, IEF-IPG, and 2D-PAGE in their performance as fractionation approaches in nano LC-ESI-MS/MS analysis of a mixture of protein standards and mitochondrial extracts isolated from rat liver. This work demonstrates that all the above techniques provide complementary protein identification results, but 1D SDS-PAGE and IEF-IPG had the highest number of identifications. The IEF-IPG technique resulted in the highest average number of detected peptides per protein. The 2D-PAGE was evaluated as a protein fractionation approach. This work shows that the recovery of proteins and resulting proteolytic digests is highly dependent on the total volume of the gel matrix. The performed comparison of the fractionation techniques demonstrates the potential of a combination of orthogonal 1D SDS-PAGE and IEF-IPG for the improved sensitivity of profiling without significant decrease in throughput.
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Project description:1. Different forms of the rat small-intestinal ;acid' beta-galactosidase were separated by using the isoelectric-focusing technique. The isoelectric points of the different forms were at pH4.2, 4.6, 5.4, 6.1 and approx. 8. 2. The two forms of ;acid' beta-galactosidase isoelectric at pH4.2 and 4.6 were completely excluded from the Sephadex G-200 gel, whereas the form isoelectric at pH8 had K(av.) 0.4. The concentration and pH of the elution buffer influenced the distribution of enzyme activity between different forms. Thus, under certain conditions of ionic strength and pH, the enzyme seems to form high-molecular-weight aggregates with low isoelectric points. These may be homopolymeric aggregates or the result of binding of enzyme to, for example, membrane fragments. The forms isoelectric at pH5.4 and 6.1 are probably aggregates of intermediate size. 3. During ion-exchange chromatography at pH6.0 one fraction of ;acid' beta-galactosidase was not retained on the column and was isoelectric at pH8 and another fraction was eluted when the buffer concentration in the eluate had increased to about 50mm. The main part of enzyme eluted in this second fraction was also isoelectric at pH8, indicating that the elution of this fraction is not a simple ion-exchange procedure but probably also involves a splitting of high-molecular-weight aggregates, originally retained because of their low isoelectric points. The enzyme subunits have a higher isoelectric point, and are therefore no longer bound to the ion-exchange resin.
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Project description:Differential measurements of sedimentation velocity showed that binding of 2 atoms of iron per molecule of human apotransferrin caused an increase in s(0) (20,w) of about 1.8%. Gel-filtration experiments to compare the elution volumes of apotransferrin and transferrin radioactively labelled with iron showed that binding of the first atom to a molecule produced a decrease in Stokes radius of about 0.7%, and the binding of a second atom an equal decrement. These results confirmed that saturation of human transferrin with iron alters the conformation sufficiently to produce detectable changes in the hydrodynamic properties. They also indicate that the local changes brought about by successive addition of 2 atoms of iron are very similar, if not identical.
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Project description:Recent advances in oxygen-tolerant controlled/living radical polymer chemistry now enable efficient synthesis of diverse and combinatorial polymer libraries. While library synthesis has been dramatically simplified, equally efficient purification strategies for removal of small-molecule impurities are not yet established in high throughput settings. It is shown that gel filtration columns for chromatography frequently used in the protein science community are well suited for high throughput polymer purification. Using either single-use columns or gel filtration plates, the purification of 32 diverse polymers is demonstrated in a library with >95% removal of small molecule impurities and >85% polymer retention in a single purification step. Doing so replaces the typical procedure of polymer precipitation, which requires solvent optimization for each polymer in a complex library. Overall, this work raises awareness in the polymer science community that gel filtration is amenable to purification of large polymer libraries and can speed up the progress of combinatorial polymer chemistry.
| S-EPMC7990394 | biostudies-literature
Project description:Unlike the case with creatinine, conditions affecting the non-glomerular filtration rate (GFR) determinants of low-molecular-weight serum proteins, ?-trace protein (BTP), ?2-microglobulin (B2M), and cystatin C, are not well characterized.Pooled cross-sectional analysis of 3 studies.3,156 persons with chronic kidney disease from the MDRD (Modification of Diet in Renal Disease) Study, AASK (African American Study of Kidney Disease and Hypertension), and CRIC (Chronic Renal Insufficiency Cohort) Study.Demographic and clinical factors hypothesized to be associated with non-GFR determinants of the filtration markers, selected from literature review and physiologic and clinical considerations.Serum creatinine, BTP, B2M, and cystatin C levels.In multivariable-adjusted errors-in-variables regression models that included adjustment for measured GFR (mGFR) and mGFR measurement error, creatinine level had stronger associations with male sex, black race, and higher urine creatinine excretion than the other filtration markers. BTP was associated less strongly with age, similar in direction with sex, and opposite in direction with race than creatinine level. Like cystatin C, B2M level was associated less strongly with age, sex, and race than creatinine level. BTP, B2M, and cystatin C levels were associated more strongly than creatinine level with other factors, including urine protein excretion and weight for BTP, smoking and urine protein excretion for B2M, and smoking for cystatin C.Findings may not be generalizable to populations without chronic kidney disease, and residual confounding with GFR due to incomplete adjustment for GFR measurement error.Like creatinine, serum levels of low-molecular-weight proteins are affected by conditions other than GFR. Knowledge of these conditions can aid the interpretation of GFR estimates and risk using these markers and guide the use of these filtration markers in developing GFR estimating equations.
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