https://www.seas.upenn.edu/%7Edischer/pdfs/Cell_on_Gel-CELLpaper.pdf

Matrix elasticity directs stem cell lineage specification.

Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells

基礎講座 電気泳動(Electrophoresis)

Alkali (Li+, Na+, K+, Rb+, and Cs+) and halide (F−, Cl−, Br−, and I−) ions play an important role in many biological phenomena, roles that range from stabilization of biomolecular structure, to influence on biomolecular dynamics, to key physiological influence on homeostasis and signaling. To properly model ionic interaction and stability in atomistic simulations of biomolecular structure, dynamics, folding, catalysis, and function, an accurate model or representation of the monovalent ions is critically necessary. A good model needs to simultaneously reproduce many properties of ions, including their structure, dynamics, solvation, and moreover both the interactions of these ions with each other in the crystal and in solution and the interactions of ions with other molecules. At present, the best force fields for biomolecules employ a simple additive, nonpolarizable, and pairwise potential for atomic interaction. In this work, we describe our efforts to build better models of the monovalent ions within t...

Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations

Ca(2+) regulation of contraction in vertebrate striated muscle is exerted primarily through effects on the thin filament, which regulate strong cross-bridge binding to actin. Structural and biochemical studies suggest that the position of tropomyosin (Tm) and troponin (Tn) on the thin filament determines the interaction of myosin with the binding sites on actin. These binding sites can be characterized as blocked (unable to bind to cross bridges), closed (able to weakly bind cross bridges), or open (able to bind cross bridges so that they subsequently isomerize to become strongly bound and release ATP hydrolysis products). Flexibility of the Tm may allow variability in actin (A) affinity for myosin along the thin filament other than through a single 7 actin:1 tropomyosin:1 troponin (A(7)TmTn) regulatory unit. Tm position on the actin filament is regulated by the occupancy of NH-terminal Ca(2+) binding sites on TnC, conformational changes resulting from Ca(2+) binding, and changes in the interactions among Tn, Tm, and actin and as well as by strong S1 binding to actin. Ca(2+) binding to TnC enhances TnC-TnI interaction, weakens TnI attachment to its binding sites on 1-2 actins of the regulatory unit, increases Tm movement over the actin surface, and exposes myosin-binding sites on actin previously blocked by Tm. Adjacent Tm are coupled in their overlap regions where Tm movement is also controlled by interactions with TnT. TnT also interacts with TnC-TnI in a Ca(2+)-dependent manner. All these interactions may vary with the different protein isoforms. The movement of Tm over the actin surface increases the "open" probability of myosin binding sites on actins so that some are in the open configuration available for myosin binding and cross-bridge isomerization to strong binding, force-producing states. In skeletal muscle, strong binding of cycling cross bridges promotes additional Tm movement. This movement effectively stabilizes Tm in the open position and allows cooperative activation of additional actins in that and possibly neighboring A(7)TmTn regulatory units. The structural and biochemical findings support the physiological observations of steady-state and transient mechanical behavior. Physiological studies suggest the following. 1) Ca(2+) binding to Tn/Tm exposes sites on actin to which myosin can bind. 2) Ca(2+) regulates the strong binding of M.ADP.P(i) to actin, which precedes the production of force (and/or shortening) and release of hydrolysis products. 3) The initial rate of force development depends mostly on the extent of Ca(2+) activation of the thin filament and myosin kinetic properties but depends little on the initial force level. 4) A small number of strongly attached cross bridges within an A(7)TmTn regulatory unit can activate the actins in one unit and perhaps those in neighboring units. This results in additional myosin binding and isomerization to strongly bound states and force production. 5) The rates of the product release steps per se (as indicated by the unloaded shortening velocity) early in shortening are largely independent of the extent of thin filament activation ([Ca(2+)]) beyond a given baseline level. However, with a greater extent of shortening, the rates depend on the activation level. 6) The cooperativity between neighboring regulatory units contributes to the activation by strong cross bridges of steady-state force but does not affect the rate of force development. 7) Strongly attached, cycling cross bridges can delay relaxation in skeletal muscle in a cooperative manner. 8) Strongly attached and cycling cross bridges can enhance Ca(2+) binding to cardiac TnC, but influence skeletal TnC to a lesser extent. 9) Different Tn subunit isoforms can modulate the cross-bridge detachment rate as shown by studies with mutant regulatory proteins in myotubes and in in vitro motility assays. (ABSTRACT TRUNCATED)

https://www.physiology.org/doi/pdf/10.1152/physrev.2000.80.2.853

Regulation of Contraction in Striated Muscle

The free solution mobility of DNA has been measured by capillary electrophoresis in the two buffers most commonly used for DNA gel electrophoresis, Tris-borate-EDTA (TBE) and Tris-acetate-EDTA (TAE). The capillaries were coated with polymers of either of two novel acrylamide monomers, N-acryloylaminoethoxyethanol or N-acryloylaminopropanol, both of which are stable at basic pH and effectively eliminate the electroendosmotic mobility due to the capillary walls. The free solution mobility of DNA in TAE buffer was found to be (3.75 +/- 0.04) x 10(-4) cm2 V-1 s-1 at 25 degrees C, independent of DNA concentration, sample size, electric field strength, and capillary coating, and in good agreement with other values in the literature. The free solution mobility was independent of DNA molecular weight from approximately 400 base pairs to 48.5 kilobase pairs, but decreased monotonically with decreasing molecular weight for smaller fragments. Surprisingly, the free solution mobility of DNA in TBE buffer was found to be (4.5 +/- 0.1) x 10(-4) cm2 V-1 s-1, about 20% larger than observed in TAE buffer, presumably because of the formation of nonspecific borate-deoxyribose complexes.

The free solution mobility of DNA.

This review describes the electrophoresis of curved and normal DNA molecules in agarose gels, polyacrylamide gels and in free solution. These studies were undertaken to clarify why curved DNA molecules migrate anomalously slowly in polyacrylamide gels but not in agarose gels. Two milestone papers are cited, in which Ferguson plots were used to estimate the effective pore size of agarose and polyacrylamide gels. Subsequent studies on the effect of the electric field on agarose and polyacrylamide gel matrices, DNA interactions with the two gel matrices, and the effect of curvature on the free solution mobility of DNA are also described. The combined results suggest that the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are primarily due to preferential interactions of curved DNAs with the polyacrylamide gel matrix; the restrictive pore size of the matrix is of lesser importance. In free solution, DNA mobilities increase with increasing molecular mass until leveling off at a plateau value of (3.17 +/- 0.01) x 10(-4) cm2/V s in 40 mM Tris-acetate-EDTA buffer at 20 degrees C. Curved DNA molecules migrate anomalously slowly in free solution as well as in polyacrylamide gels, explaining why the Ferguson plots of curved and normal DNAs containing the same number of base pairs extrapolate to different mobilities at zero gel concentration.

/pdf/electrophoresis-of-dna-in-agarose-gels-polyacrylamide-gels-1miq0bzdsr.pdf

Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution

DNA restriction fragments ranging from 79 to 789 base pairs in length have been characterized by transient electric birefringence (TEB) measurements at various temperatures between 4 and 43°C The DNA fragments do not contain runs of four or more adenine residues in a row and migrate with normal electrophoretic mobilities in polyacrylamide gels, indicating that they are not intrinsically curved or bent The low ionic strength buffers used for the measurements contained 1 mM Tris Cl, pH 80, EDTA, and variable concentrations of Na+ or Mg2+ ions The rotational relaxation times were obtained by fitting the TEB field-free decay signals with a nonlinear least-squared fitting program; the decay of the birefringence was monoexponential for fragments ≤ 241 base pair (bp) in length and multiexponential for larger fragments The terminal relaxation times, characteristic of the end-over-end rotation of the DNA molecules, were then used todetermine the persistence length (p) and hydrodynamic radius (r) of DNA as a function of temperature and ionic strength, using several different hydrodynamic models The specific values obtained for p and r are model dependent The wormlike chain model of P J Hagerman and B H Zimm (Biopolymers 1981, Vol 20, pp 1481–1502) combined with the revised Broersma equation (J Newman et al, Journal of Mol Biol 1997, Vol 116, pp 593–606) appears to be the most suitable for describing the flexibility of DNA in low ionic strength solutions The values of p and r obtained from the global least squares fitting of this equation are independent of DNA length, and the deviations of the individual values from the average are reasonably small The consensus r value calculated for DNA in various low ionic strength solutions containing 1 mM Tris buffer is 147 ± 04 A at 20°C The consensus p values decrease from 814 ∼ 564 A in solutions containing 1 mM Tris buffer plus 02–1 mM NaCl and decrease still further to 440 A in solutions containing 02 mM Mg2+ ions The persistence length exhibits a shallow maximum at 20°C and decreases slowly upon either increasing or decreasing the temperature, regardless of the model used to fit the data By contrast, the consensus values of the hydrodynamic radius are independent of temperature The calculated persistence lengths and hydrodynamic radii are compared with other data in the literature © 2002 Wiley Periodicals, Inc Biopolymers 61: 261–275, 2002

DNA persistence length revisited

The electrophoretic mobilities and diffusion coefficients of single- and double-stranded DNA molecules up to 50 000 bases or base pairs in size have been analyzed, using mobilities and diffusion coefficients either measured by capillary electrophoresis or taken from the literature. The Einstein equation suggests that the electrophoretic mobilities (μ) and diffusion coefficients (D) should be related by the expression μ/D = Q/kBT, where Q is the charge of the polyion (Q = zeo, where z is the number of charged residues and eo is the fundamental electronic charge), kB is Boltzmann's constant, and T is the absolute temperature. If this equation were true, the ratio μ/zD should be a constant equal to eo/kBT (39.6 V-1) at 20 °C. However, the ratio μ/zD decreases with an increase in molecular weight for both single- and double-stranded DNAs. The mobilities and diffusion coefficients are better described by the modified Einstein equation μ/NmD = eo/kBT, where N is the number of repeat units (bases or base pairs) ...

Unified description of electrophoresis and diffusion for DNA and other polyions.

The mobilities of various DNA fragments in two normally migrating molecular weight ladders were studied in polyacrylamide gels containing different concentrations of the crosslinker N,N'-methylenebisacrylamide (Bis). The acrylamide concentration ranged from 2.5-10.5%T (w/v); the Bis concentration ranged from 0.5-10%C (w/w), with respect to total acrylamide. Ferguson plots were constructed for each of the DNA fragments in gels of each composition. The Ferguson plots of the different multimers in each molecular weight ladder were nearly parallel in gels containing 0.5-3%C, converged close to a common intercept at zero gel concentration in gels containing 4%C, and crossed at approximately 1.5%T in gels containing 5 and 10%C. If the mobilities observed for the different DNA fragments at zero gel concentration were also extrapolated to zero DNA molecular weight, a common limiting mobility was observed in gels of all crosslinker concentrations. This limiting mobility was approximately equal to the free solution mobility of DNA. The effective pore radius of each gel was estimated from Ferguson plots based on relative mobilities, using the mobility of the smallest DNA fragment in each molecular weight ladder as the reference mobility. The calculated gel pore radii ranged from 142 nm to 19 nm, respectively, for gels containing 4.6%T, 1.5%C, and 10.5%T, 5 or 10%C. These pore radii are an order of magnitude larger than previously accepted values, but are consistent with scanning electron microscope measurements (Ruchel, R., et al., J. Chromatogr. 1978, 42, 77-90).(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of polyacrylamide gel pore size from Ferguson plots of linear DNA fragments. II. Comparison of gels with different crosslinker concentrations, added agarose and added linear polyacrylamide.

Nancy C. Stellwagen

Papers

The free solution mobility of DNA.

Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution

DNA persistence length revisited

Unified description of electrophoresis and diffusion for DNA and other polyions.

Estimation of polyacrylamide gel pore size from Ferguson plots of linear DNA fragments. II. Comparison of gels with different crosslinker concentrations, added agarose and added linear polyacrylamide.