What Does Using A Dna Size Standard Accomplish?
Electrophoresis. Author manuscript; available in PMC 2019 Jan 30.
Published in final edited course every bit:
PMCID: PMC6352729
NIHMSID: NIHMS1002113
Deoxyribonucleic acid ladders can be used to size polyphosphate resolved by polyacrylamide gel electrophoresis
Stephanie A. Smith
iSection of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA
Yan Wang
2Current address: Bayer Pharmaceuticals, San Francisco, CA
James H. Morrissey
1Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, The states
Abstract
Folio is oft used to resolve inorganic polyphosphates (polyP), just unfortunately polyP size ladders are not commercially available. Since several dyes that are unremarkably used to detect nucleic acids in gels also stain polyP, we examined the utility of commercially bachelor Dna size ladders for estimating polyP polymer lengths by gel electrophoresis. Narrow size fractions of polyP were prepared and their polymer lengths were quantified using NMR. Commercially available Dna ladders and these polyP fractions were then subjected to PAGE to determine the human relationship between migration of Deoxyribonucleic acid vs polyP, which was found to be: log10(dsDNA length in bp) = i.66 × logten(polyP length in phosphate units) − ane.97. This human relationship between DNA and polyP size held for a variety of different polyacrylamide concentrations, indicating that Deoxyribonucleic acid size ladders tin can readily be employed to estimate polyP polymer lengths by Folio.
Keywords: Deoxyribonucleic acid ladder, Electrophoresis, Polyphosphate, Folio
1. Introduction
Inorganic polyphosphates (polyP) are linear polymers of orthophosphate residues that are widespread throughout biology [1]. Although studied nigh intensively in unicellular organisms [i, 2], functions for polyP in higher organisms are at present being revealed [three, 4]. This includes roles in blood clotting [5], angiogenesis [half-dozen], apoptosis [7], jail cell proliferation [8], free energy metabolism [ix], bone mineralization [10, xi], and tumor metastasis [12]. PolyP tin can also be non-enzymatically covalently attached to lysine residues in some proteins. Since polyP varies profoundly in size depending on the source, and since the biological activities of polyP depend markedly on polymer length [13], it can be important to determine the size of polyP isolated from biological sources.
While methods other than PAGE exist for quantifying polyP polymer size, they are laborious and slow. Page is therefore a popular method for resolving polyP and determining the distribution of its polymer lengths [fourteen, fifteen]. Although nosotros have successfully employed narrowly size-fractionated polyP preparations for use as PAGE sizing ladders [13], polyP fractions of well-divers sizes are labor-intensive to prepare and not commercially available. We hypothesized that commercially available DNA ladders could be used as convenient size markers for estimating polyP polymer lengths using Page, especially since both polyP and DNA are linear, anionic polymers that tin be stained using dyes such equally toluidine blue or 4′,6-diamidino-2-phenylindole (DAPI) [14, xvi]. We therefore investigated the relationship between migration of polyP and Deoxyribonucleic acid on PAGE. We now report conditions under which Dna size ladders can routinely be used to estimate polyP polymer lengths via PAGE.
2. Materials and methods
ii.1. Reagents
All chemicals were of analytical form from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Fair Lawn, NJ). The 10 bp DNA ladder was from Life Technologies (Grand Island, NY), and the 100 bp Deoxyribonucleic acid ladder was from New England Biolabs (Ipswich, MA). Heterogeneous polyP preparations of varying size ranges were purchased from Sigma-Aldrich or were a kind gift from BK Giulini GmbH (Ludwigshafen, Germany).
2.two. PolyP fractionation past preparative Page
Starting from heterogeneous polyP preparations, preparative Page was used to isolate narrowly size-fractionated polyP as previously described [13, xvi]. PolyP concentrations were quantified by measuring inorganic phosphate [12] following hydrolysis in 1 1000 HCl at 100°C for 10 min, every bit described [sixteen]. PolyP concentrations are reported here as phosphate monomer concentration.
2.3. Sizing of polyP via solution NMR
Eight narrowly size-fractionated polyP preparations were employed as size standards in this report. An aliquot of each was lyophilized and then dissolved in water containing 10% (v/v) D2O, after which the pH was adapted to nine. One-dimensional 31P spectra of polyP samples were acquired three times at 23°C with a 3 sec recycle delay on a Varian Unity INOVA600 spectrometer with a 5 mm AutoTuneX 1H/X PFG Z probe. Chemical shifts were externally referenced with phosphoric acid at 0 ppm. Spectra were point-averaged until the blastoff phosphate summit achieved a indicate-to-noise ratio of at least v:1. All spectra were processed using MNOVA (MestreLab Research) with baseline correction and i Hz line broadening. An case of such a spectrum is shown in Supporting Fig. 1. Integration was performed to quantify the acme areas. The length of each polyP polymer was calculated from the ratios of the integration values for the blastoff (terminal) phosphates and the internal phosphates, as described [13]. The polymer lengths of the polyP preparations adamant using NMR are detailed in Supporting Table 1.
2.4. Belittling gel electrophoresis of polyP
PolyP was resolved by PAGE using commercially bachelor, eight.half-dozen × 6.8 cm polyacrylamide TBE gels from Bio-Rad (Hercules, CA), containing fixed 5%, 10% or fifteen% polyphosphate, or a gradient of iv–20% polyacrylamide. Running buffer was TBE, and 5× sample buffer independent 5× TBE, 15% Ficoll 400, and 0.1% bromophenol bluish. PolyP samples were loaded onto gels at ten nmol phosphate monomer per lane. Electrophoresis was performed at 150 V for xx to fifty min at 25° C. Gels were stained with agitation for ten min with 0.05% toluidine blueish in 25% methanol/5% glycerol, destained in iii changes of 25% methanol/5% glycerol over three hr, and imaged in white light on a GelDoc XR (Bio-Rad).
2.5. Evaluation of DNA ladder migration
Page migration distances for bands in commercially available Deoxyribonucleic acid ladders were analyzed using ImageJ software [17]. Migration distances were determined by identifying the pinnacle intensity of each band using the "plot lane" feature of the software. These distances were converted to Rf values by dividing the migration distance of the ring past that of bromophenol blue. A standard curve was created by plotting logx of the number of bp in the DNA polymer (y-axis) vs Rf (x-axis) using Sigma Plot 12.5. A cubic polynomial was fitted to the data set of identifiable DNA bands to achieve an rii of >0.99.
3. Results and give-and-take
The goal of these studies was to develop a uncomplicated, reproducible Page-based method for estimating polyP polymer lengths using commercially available DNA ladders.
3.1. Cosmos of the Deoxyribonucleic acid equivalence model using 4–xx% gels
Commercial Deoxyribonucleic acid ladders and eight polyP preparations whose polymer lengths had been divers by NMR (ranging from 14 to 617 phosphates long) were resolved by Folio in six separate experiments on TBE gels containing a slope of 4–20% polyacrylamide. The Rf values for the Deoxyribonucleic acid and polyP bands were measured relative to migration of the bromophenol blueish dye. For the bands in the DNA ladders, the log10(DNA length) was plotted vs Rf, to which a cubic polynomial was fitted:
Equation 1:
A representative example of a iv–20% gel is shown in Fig. 1A. The associated plot of DNA length vs Rf, along with the fitted polynomial, is shown in Fig. 1B.
Representative comparison of the electrophoretic mobilities of Deoxyribonucleic acid ladders and size-fractionated polyP preparations used to create the Deoxyribonucleic acid equivalence model. (A) DNA ladders and polyP samples were resolved on 4–20% polyacrylamide gels. PolyP polymer lengths as determined past NMR are indicated at the top of the lanes. Deoxyribonucleic acid ladders in bp (provided by the manufacturer) are indicated at left (for the x bp ladder) or right (for the 100 bp ladder). "BPB" indicates the migration of the bromophenol blueish tracking dye. (B) Relationship betwixt DNA length and migration on PAGE. Log10 of Deoxyribonucleic acid lengths in bp for individual bands in the 10 bp (△) and 100 bp Deoxyribonucleic acid ladders (○) were plotted against Rf. A cubic polynomial (solid line) was fitted to the data.
For each individual 4–20% gel, the fitted polynomial was used to convert the Rf value of each polyP band into the equivalent Dna length predicted to migrate at that Rf value. These predicted Deoxyribonucleic acid length equivalents were then used to gear up the migration equivalence model as described below. Notation that the smallest polyP polymer (14 phosphates) stained poorly with toluidine blue and sometimes migrated off the gel, so this polyP ring was excluded from further analysis. The two next smallest polyP polymers (44 and 50 phosphates) migrated faster than the smallest available Deoxyribonucleic acid ring (ten bp), and then these polyP polymers were also excluded from developing the migration equivalence model.
To generate the Dna equivalence model, nosotros used the polynomial derived from migration of bands in the DNA ladders to calculate the predicted equivalent Dna lengths in bp for the remaining polyP bands (81, 129, 288, 390, and 617 phosphates long) resolved on 4–20% gels. These equivalent DNA lengths are plotted vs polyP length as open circles in Fig. ii. A line was fitted to these data, yielding an equivalence model of:
Equation ii:
This data set therefore describes the equivalence between polyP length determined from NMR and calculated DNA length based on Rf values on PAGE for 4–twenty% gels. Note that although the polyP polymers containing 44 or 50 phosphates were not used to create the equivalence model, their extrapolated Dna equivalence values (open diamonds in Fig. ii) fell on the aforementioned line as the equivalence information for the five larger polyP standards.
Deoxyribonucleic acid-polyP equivalence model. Commercial Deoxyribonucleic acid ladders and size-fractionated polyP preparations were resolved on six carve up 4–20% polyacrylamide gels and analyzed as described in the text. The calculated equivalent Deoxyribonucleic acid lengths (in bp) for the polyP bands in all six gels are plotted vs. the NMR-determined polyP lengths (in phosphates) on logten-logx axes. Plotted as open circles are the five polyP preparations used to create the model, which ranged in size from 81 to 617 phosphates. A line was fitted to these data (rtwo = 0.99), yielding the equivalence model given in the text as Equation 2. 2 of the polyP preparations (44 and 50 phosphate units long, ◇) migrated faster than the smallest band in the Dna ladder, which was 10 bp. Although these 2 polyP sizes were not used to set up the equivalence model, their values notwithstanding prevarication essentially on the aforementioned line.
three.two. Awarding of the Dna equivalence model to gels of other polyacrylamide concentrations
We next evaluated whether the Dna equivalence model could be used to estimate the lengths of polyP resolved on not-gradient TBE gels containing five%, 10%, or 15% polyacrylamide. Representative examples of these gels are presented in Fig. 3. Equally with the 4–20% gels, the logten(DNA length) values for the Dna ladder bands were plotted vs Rf, to which cubic polynomials were fitted. For each gel, the polynomial was used to catechumen Rf values of the polyP bands into equivalent predicted Deoxyribonucleic acid sizes in bp, as described above for the 4–20% gels. PolyP sizes were and then calculated according to the following rearrangement of equation 2:
Equation 3:
The operation of this model using v%, 10%, and xv% polyacrylamide gels is shown in Fig. 4A. The agreement between measured and calculated polyP lengths was excellent, with a Pearson Correlation coefficient of 0.982. A bias in performance of the model relative to the polyacrylamide concentration was not apparent from this plot. This finding indicates that the relationship described in Equations 2 and 3, although developed using four–20% gradient gels, applies to non-gradient gels over a range of polyacrylamide concentrations.
Representative comparisons of the electrophoretic mobilities of Deoxyribonucleic acid ladders and size-fractionated polyP preparations resolved on gels with stock-still percentages of polyacrylamide. DNA ladders and polyP samples were resolved on (A) 15%; (C) 10%; or (E) 5% polyacrylamide gels. PolyP polymer lengths as determined by NMR are indicated at the summit of the lanes. DNA ladders in bp (provided past the manufacturer) are indicated at left (10 bp ladder) or right (100 bp ladder). "BPB" indicates the migration of the bromophenol blue tracking dye. Logx of DNA lengths in bp for individual bands in the x bp (△) and 100 bp Deoxyribonucleic acid ladders (○) were plotted against Rf for: (B) 15%; (D) 10%; or (F) v% polyacrylamide gels. Cubic polynomials (solid lines) were fitted to the data.
Operation of the DNA equivalence model. (A) Awarding of the equivalence model to judge polyP lengths using various gels. PolyP standards of known lengths (determined by NMR) and bands in x bp or 100 bp DNA ladders were resolved on v% (○), 10% (□), or fifteen% (▽) polyacrylamide gels (4 each) as visualized in Fig. three. Predicted polymer length of each polyP ring was calculated by reference to migration of Deoxyribonucleic acid ladders resolved on the same gel, equally described in the text. These predicted polyP lengths are plotted here vs. their lengths adamant by NMR, with the dotted line representing equivalence between predicted and measured lengths. Concurrence between measured and predicted polyP polymer sizes had a Pearson correlation coefficient of 0.982. (B) Reproducibility of the polyP sizing method. Thirteen narrowly size-fractionated polyP samples ("A" through "Yard" on the x-axis), just of undetermined length, were resolved together with commercial Deoxyribonucleic acid ladders on v% (○), x% (□), 15% (▽) or iv–twenty% (△) polyacrylamide gels (four gels each). Polymer lengths of the polyP bands were predicted from the polynomial fit of the migration of the Dna ladder and the equivalence model as defined in Fig. 3. The resulting (calculated) polyP polymer lengths using this method are plotted on the y-centrality.
3.iii. Reproducibility of polyP size values obtained using the DNA equivalence model
Using thirteen additional size-fractionated polyP preparations of unknown polymer lengths, we resolved the preparations on 5%, 10%, 15%, and 4–twenty% polyacrylamide gels and evaluated the inter-analysis variability for determining their polymer lengths using the model. The sizes of these polyP samples estimated using our DNA equivalence model are presented in Fig. 4B, with a mean coefficient of variation of 0.08 ± 0.03 (± standard deviation)
4. Concluding remarks
Advances in agreement the physiologic roles for polyP in mammalian biology [18] crave more sensitive methods to notice, characterize, and quantify inorganic polyP. The method described hither for estimating the polymer lengths of polyP via PAGE on TBE gels using commercially available Deoxyribonucleic acid ladders is simple, easily achieved, inexpensive, and reproducible. The method performed equivalently well on gels containing a range of unlike polyacrylamide concentrations, including gradient and not-gradient gels. While our model was reliable for sizing polyP longer than 62 units (the size that comigrated with the smallest DNA ring), the lack of bachelor dsDNA standards <10bp makes it difficult to precisely evaluate polyP of smaller size.
In that location are many automated systems bachelor for analyzing Page information using dsDNA size ladders resolved on the same gel to catechumen the migration data of unknown bands into DNA lengths in bp. Such automated gel analysis systems should be readily usable in the method described in this paper. Thus, once the migration data of polyP bands have been converted into "bp" from the automated assay, these bp values can be farther converted into polyP sizes (in phosphate units) using Equation 3. Alternatively, if the equivalent polyP length for each DNA band is input into gel evaluation software, and then the sizes calculated by the software for unknown polyP bands will be output directly in phosphate units. For the reader'due south convenience, the polyP length equivalents for several mutual Dna ladder bands are presented in Tabular array 1.
Table one.
PolyP lengths represented past common DNA ladder bands
| DNA (bp) | Equivalent polyP length (phosphates) |
|---|---|
| 10 | 62 |
| 25 | 107 |
| 50 | 162 |
| 75 | 207 |
| 100 | 246 |
| 200 | 374 |
| 300 | 478 |
| 400 | 568 |
| 500 | 650 |
| 1000 | 986 |
Supplementary Cloth
supplement
Acknowledgements
The authors thank Sulalita Chaki for technical assistance. These studies were supported by grants R35 HL135823 and UM1 HL120877 from the National Eye, Lung and Blood Institute of the NIH.
Abbreviations:
| DAPI | 4',6-diamidino-two-phenylindole |
| polyP | inorganic polyphosphate |
Footnotes
S.A.S. and J.H.M. are co-inventors on patents and pending patent applications on medical uses of polyP and polyP inhibitors. Y.W. declares no conflicting fiscal interests.
5 References
[1] Kornberg A, Rao NN, Ault-Riché D, Annu. Rev. Biochem 1999, 68, 89–125. [PubMed] [Google Scholar]
[2] Kulaev IS, Kulakovskaya TV, Andreeva NA, Lichko LP, Prog. Mol. Subcell. Biol 1999, 23, 27–43. [PubMed] [Google Scholar]
[4] Schröder HC, Lorenz B, Kurz L, Muller WE, Prog. Mol. Subcell. Biol 1999, 23, 45–81. [PubMed] [Google Scholar]
[5] Smith SA, Mutch NJ, Baskar D, Rohloff P, Docampo R, Morrissey JH, Proc. Natl. Acad. Sci. U. S. A 2006, 103, 903–908. [PMC gratis article] [PubMed] [Google Scholar]
[7] Hernandez-Ruiz Fifty, González-García I, Castro C, Brieva JA, Ruiz FA, Haematologica 2006, 91, 1180–1186. [PubMed] [Google Scholar]
[8] Wang 50, Fraley CD, Faridi J, Kornberg A, Roth RA, Proc. Natl. Acad. Sci. U. S. A 2003, 100, 11249–11254. [PMC costless article] [PubMed] [Google Scholar]
[nine] Pavlov E, Aschar-Sobbi R, Campanella K, Turner RJ, Gómez-García MR, Abramov AY, J. Biol. Chem 2010, 285, 9420–9428. [PMC free article] [PubMed] [Google Scholar]
[x] Schröder HC, Kurz 50, Müller WE, Lorenz B, Biochemistry (Mosc). 2000, 65, 296–303. [PubMed] [Google Scholar]
[11] Leyhausen Yard, Lorenz B, Zhu H, Geurtsen Westward, Bohnensack R, Müller Nosotros, Schröder HC, J. Os Miner. Res 1998, xiii, 803–812. [PubMed] [Google Scholar]
[12] Tammenkoski M, Koivula K, Cusanelli Due east, Zollo Chiliad, Steegborn C, Baykov AA, Lahti R, Biochemistry 2008, 47, 9707–9713. [PubMed] [Google Scholar]
[xiii] Smith SA, Choi SH, Davis-Harrison R, Huyck J, Boettcher J, Reinstra CM, Morrissey JH, Blood 2010, 116, 4353–4359. [PMC free article] [PubMed] [Google Scholar]
[fourteen] Lorenz B, Schröder HC, Prog. Mol. Subcell. Biol 1999, 23, 217–239. [PubMed] [Google Scholar]
[xv] Robinson NA, Goss NH, Wood HG, Biochem. Int 1984, 8, 757–769. [PubMed] [Google Scholar]
[sixteen] Smith SA, Morrissey JH, Electrophoresis 2007, 28, 3461–3465. [PubMed] [Google Scholar]
What Does Using A Dna Size Standard Accomplish?,
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