Methodological aspects of gel filtration with special reference to desalting operations

Flodin, Per

doi:10.1016/s0021-9673(01)92827-4

Cited by 301 publications

(51 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This gives the experimenter a stock of sample aliquots which may be manipulated at will and then passed through a second column to determine the effect of the manipulation on the release of tritium. As has been shown by Flodin (1960, Fig. 4B), a sufficiently large sample will yield effluent diluted hardly at all but with free tritium reduced by more than 100-fold.…”

Section: The Separationmentioning

confidence: 57%

A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease^*

Sw¹

1963

Biochemistry

190

View full text Add to dashboard Cite

A new method for measuring the hydrogen exchange of macromolecules in solution is described. The method uses tritium to trace the movement of hydrogen, and utilizes Sephadex columns to effect, in about 2 minutes, a separation between tritiated macromolecule and tritiated solvent great enough to allow the measurement of bound tritium. High sensitivity and freedom from artifact is demonstrated and the possible value of the technique for investigation of other kinds of colloidsmall molecule interaction is indicated. Competition experiments involving tritium, hydrogen, and deuterium indicate the absence of any equilibrium isotope effect in the ribonuclease-hydrogen isotope system, though a secondary kinetic isotope effect is apparent when ribonuclease is largely deuterated. Ribonuclease shows four clearly distinguishable kinetic classes of exchangeable hydrogens. Evidence is marshaled to suggest the independently measurable classes II, III, and IV (in order of decreasing rate of exchange) to represent "random-chain" peptides, peptides involved in α-helix, and otherwise shielded side-chain and peptide hydrogens, respectively.Protons attached to nitrogen, oxygen, and sulfur of molecules dispersed in liquid solution are, in general, able to exchange with mobile protons in the solvent. The rate of exchange of protons occupying specific molecular sites may be modified, or even largely controlled, by the details of molecular architecture in their immediate vicinity. Thus measurement of the kinetics of the hydrogen exchange of macromolecules can provide a fine probe for identifying and quantitating details of conformation and of changes in conformation of macromolecules.A practicable method for the measurement of hydrogen-exchange kinetics was first developed and used in the study of structure in proteins and polypeptides by Linderstrøm-Lang and his co-workers . Typically, in this method, deuterated protein is separated from deuterated solvent by freeze-drying, the protein is redissolved in water, and the kinetics of the loss of deuterium by the protein is followed by analyzing samples of solvent, after various times of protein incubation, for deuterium content. Deuterium content is determined by density measurement of tiny samples of solvent isolated from the protein by a second freeze-drying step. The method, though ingenious in conception, seems unusually demanding in execution and makes use of procedures which might be considered questionable. While the method is capable of considerable internal precision, a number of the operations involved, freezing, drying, heating (60°), redissolution,

show abstract

Section: The Separationmentioning

confidence: 57%

A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease^*

Sw¹

1963

Biochemistry

190

View full text Add to dashboard Cite

show abstract

“…A 50-ml sample of fetal calf serum was applied to the column and desalted according to the procedure of Flodin (17). The serum proteins were recovered in the first portion of the effluent and all the low molecular weight constituents were recovered from 400 ml of the remaining effluent.…”

Section: Separated By Gel Filtrationmentioning

confidence: 99%

Cultivation of Mammalian Cells in Defined Media With Protein and Nonprotein Supplements

Healy

Parker

1966

The Journal of Cell Biology

View full text Add to dashboard Cite

An improved chemically defined basal medium (CMRL-1415) has been used to advantage in studying the effects on trypsinized, newly explanted mouse embryo cells of certain glycoproteins from plasma and serum, certain nonprotein macromolecules, and various combinations of these, in stationary cultures. When protein and nonprotein fractions were separated from fetal calf serum, the entire growth activity was found to be associated with the protein. When 100 mg % of dialyzed, freeze-dried, supernatant solution of Cohn's fraction V (method 6) from human plasma was used as a supplement for CMRL-1415, there was considerable improvement in the cultures; and seromucoid prepared from calf serum had a similar effect. Supernatant V was further fractionated by gel filtration to give a threefold concentration of growth activity in a single, highly purified o~x-acid glycoprotein (orosomucoid). Starch gel electrophoresis of horse serum that was used to supplement the basal medium revealed a decrease of both oq-acid glycoprotein and ~-macroglobulin during the cultivation of mouse embryo cells. When horse serum was fractionated on DEAEcellulose columns, the only fraction that showed growth activity was a slow as-globulin. When the ~2-macroglobulin of Schultze was prepared from horse serum by salt precipitation, it was equally effective. When the ~2-macroglobulin from horse serum was tested (at 100 mg %) in combination with otl-acid glycoprotein from Supernatant V, seromucoid from calf serum, or unfractionated Supernatant V, the growth response was greatly in excess of that produced by any of these supplements tested separately. The ~2-macroglobufin from horse serum could be replaced by certain nonprotein macromolecules (e.g., dextran or Ficoll). ~hus, dextran (mol. wt. 100,000 to 200,000) had no visible effect on the cells when used alone at 0.1 or 1%. But when these levels of dextran were used in combination with low molecular weight glycoproteins (e.g., unfractionated Supernatant V at 100 mg %), the cultures remained active and healthy for unusually long periods.

show abstract

“…Experimental as well as theoretical investigations led to the establishment of a method to estimate the distribution coefficienU- 3 ) The second is the dispersion of the elution curve (peak spreading, zone broadening). [4][5][6][7][8][9][10][11][12][13][14] The degree of dispersion in a column plays a more important role in gel chromatography, since it directly affects the separation efficiency and the degree of dilution of the sample.…”

mentioning

confidence: 99%

Analysis of Dispersion Mechanism in Gel Chromatography

Nakanishi

Yamamoto

Matsuno

et al. 1977

Agricultural and Biological Chemistry

View full text Add to dashboard Cite

Methodological aspects of gel filtration with special reference to desalting operations

Cited by 301 publications

References 15 publications

A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease^*

A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease^*

Cultivation of Mammalian Cells in Defined Media With Protein and Nonprotein Supplements

Analysis of Dispersion Mechanism in Gel Chromatography

Contact Info

Product

Resources

About

Methodological aspects of gel filtration with special reference to desalting operations

Cited by 301 publications

References 15 publications

A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease*

A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease*

Cultivation of Mammalian Cells in Defined Media With Protein and Nonprotein Supplements

Analysis of Dispersion Mechanism in Gel Chromatography

Contact Info

Product

Resources

About

A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease^*

A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease^*