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

Abstract

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 (Rüchel, R., et al., J. Chromatogr. 1978, 42, 77-90).(ABSTRACT TRUNCATED AT 250 WORDS)

Frozen Acrylamide Gels as Dynamic Nuclear Polarization Matrices

“…Varying the monomer to cross linker ratio has the known effect of varying the interstitial space in the gel, which can be thought of in terms of a pore size. [23] In order to remove excess initiators (that can react with the polarizing agents) and residual monomers, the gel is then purified using the breathing technique of Willner and coworkers (see SI), [24] initially developed to uniformly distribute gold NPs in the gel matrix. Here we first add acetone to the gel, which collapses and turns as a white soft solid (see Figure S2 (b)) as the water containing APS and TEMED is expulsed.…”

“…Here the pore size decreases from gel A>B>C, and varies between 41 to 10 nm, respectively, as estimated using the approach of Holmes et al). [23] Thus we can hypothesise that smaller pores improve either the homogeneous dispersion of the free radical in the gel, or the quality of the glass formed upon cooling, and leads to better DNP enhancements.…”

Highly Sensitive Methods Using Liquid Chromatography and Capillary Electrophoresis for Quantitative Analysis of Glycoprotein Glycans

“…The pore size of this acrylamide gel is unknown. However, the respective pore sizes of 4.6%T/1.5%C and 10.5%T/10%C polyacrylamide gels are reportedly 142 and 19 nm [69]. Therefore, the pore size of the gel used for the study described herein is estimated as less than 10 nm, which is sufficient to obtain permselectivity.…”

Spatially resolved microrheology of heterogeneous biopolymer hydrogels using covalently bound microspheres

“…Polyacrylamide gel, with pore size smaller that the microsphere size, was used as it is known to be more homogeneous compared to natural biopolymers, thereby allowing us to compare the MSD distributions between NHS-microspheres and COOHmicrospheres in the absence of heterogeneities that complicate the distribution measurements. Polyacrylamide gels with total monomer concentrations of 10.5 (w/v) %T and 4.6 (w/v) %T have estimated median pore radii of 70 nm and 107 nm, respectively (Holmes and Stellwagen 1991). Red fluorescent NHS-microspheres and green fluorescent COOH-microspheres, both 0.2 µm in size, were co-dispersed in each polyacrylamide gel sample to circumvent the problem of batch-to-batch sample variability, from preparing the …”