Polysaccharide as a Separation Medium for Gel Electrophoresis
<p>Comparison of size exclusion chromatography and gel electrophoresis. (<b>A</b>) SEC, (<b>B</b>) gel electrophoresis. The size of the arrow indicates the mobility of the macromolecule. In SEC, a larger macromolecule moves faster, while in electrophoresis, it moves slower. The migration positions in the SEC column and electrophoresis are shown in each panel.</p> "> Figure 2
<p>Comparison of gel structures. (<b>A</b>) Agarose, (<b>B</b>) polyacrylamide, (<b>C</b>) starch. This is an original diagram drawn by us with reference to refs. [<a href="#B9-polysaccharides-05-00024" class="html-bibr">9</a>,<a href="#B10-polysaccharides-05-00024" class="html-bibr">10</a>,<a href="#B11-polysaccharides-05-00024" class="html-bibr">11</a>,<a href="#B12-polysaccharides-05-00024" class="html-bibr">12</a>,<a href="#B13-polysaccharides-05-00024" class="html-bibr">13</a>,<a href="#B14-polysaccharides-05-00024" class="html-bibr">14</a>,<a href="#B15-polysaccharides-05-00024" class="html-bibr">15</a>,<a href="#B16-polysaccharides-05-00024" class="html-bibr">16</a>].</p> "> Figure 3
<p>Comparison of protein and agarose gel. (<b>A</b>) Protein gel is assumed to possess a negative charge (−). (<b>B</b>) Agarose gel is assumed to possess no charge.</p> "> Figure 4
<p>Relation between the running pH of the gel electrophoresis and protein/nucleic acid charges. The running pH is set at pH 6.1.</p> "> Figure 5
<p>The effects of gel pore size on the state of protein (BSA). Small cell effects lead to oligomerization (<b>upper panel</b>) or protein folding (<b>lower panel</b>).</p> "> Figure 6
<p>Excluded volume of protein.</p> "> Figure 7
<p>Digestion pattern of three plasmids by restriction enzyme. (<b>A</b>) Different formats of gel. (<b>B</b>) Digested plasmids were analyzed by agarose native gel electrophoresis. Digital image of 3 plasmid restriction digests run on a 1% agarose gel using TAE buffer, stained with fluorescence dye (GelGreen). The DNA size marker is a commercial ladder. The positions of the wells and directions of DNA migration are noted. These are our original data.</p> "> Figure 8
<p>Schematic illustration of gel shift assay with pH 8.3 TAE (<b>A</b>) or pH 6.1 His/MES (<b>B</b>) buffer systems. Nucleic acid is titrated with a presumably very basic protein, whereas a better resolution of the complex may be expected for the pH 6.1 system. This is an original diagram drawn by us.</p> "> Figure 9
<p>Comet assay. (<b>A</b>) Experimental scheme. (<b>B</b>) Typical data with intact and degraded/damaged DNA. This is an original diagram drawn by us with reference to refs. [<a href="#B37-polysaccharides-05-00024" class="html-bibr">37</a>,<a href="#B38-polysaccharides-05-00024" class="html-bibr">38</a>,<a href="#B39-polysaccharides-05-00024" class="html-bibr">39</a>,<a href="#B40-polysaccharides-05-00024" class="html-bibr">40</a>,<a href="#B41-polysaccharides-05-00024" class="html-bibr">41</a>,<a href="#B42-polysaccharides-05-00024" class="html-bibr">42</a>,<a href="#B43-polysaccharides-05-00024" class="html-bibr">43</a>,<a href="#B44-polysaccharides-05-00024" class="html-bibr">44</a>,<a href="#B45-polysaccharides-05-00024" class="html-bibr">45</a>,<a href="#B46-polysaccharides-05-00024" class="html-bibr">46</a>,<a href="#B47-polysaccharides-05-00024" class="html-bibr">47</a>,<a href="#B48-polysaccharides-05-00024" class="html-bibr">48</a>,<a href="#B49-polysaccharides-05-00024" class="html-bibr">49</a>,<a href="#B50-polysaccharides-05-00024" class="html-bibr">50</a>,<a href="#B51-polysaccharides-05-00024" class="html-bibr">51</a>,<a href="#B52-polysaccharides-05-00024" class="html-bibr">52</a>,<a href="#B53-polysaccharides-05-00024" class="html-bibr">53</a>,<a href="#B54-polysaccharides-05-00024" class="html-bibr">54</a>,<a href="#B55-polysaccharides-05-00024" class="html-bibr">55</a>,<a href="#B56-polysaccharides-05-00024" class="html-bibr">56</a>,<a href="#B57-polysaccharides-05-00024" class="html-bibr">57</a>,<a href="#B58-polysaccharides-05-00024" class="html-bibr">58</a>].</p> "> Figure 9 Cont.
<p>Comet assay. (<b>A</b>) Experimental scheme. (<b>B</b>) Typical data with intact and degraded/damaged DNA. This is an original diagram drawn by us with reference to refs. [<a href="#B37-polysaccharides-05-00024" class="html-bibr">37</a>,<a href="#B38-polysaccharides-05-00024" class="html-bibr">38</a>,<a href="#B39-polysaccharides-05-00024" class="html-bibr">39</a>,<a href="#B40-polysaccharides-05-00024" class="html-bibr">40</a>,<a href="#B41-polysaccharides-05-00024" class="html-bibr">41</a>,<a href="#B42-polysaccharides-05-00024" class="html-bibr">42</a>,<a href="#B43-polysaccharides-05-00024" class="html-bibr">43</a>,<a href="#B44-polysaccharides-05-00024" class="html-bibr">44</a>,<a href="#B45-polysaccharides-05-00024" class="html-bibr">45</a>,<a href="#B46-polysaccharides-05-00024" class="html-bibr">46</a>,<a href="#B47-polysaccharides-05-00024" class="html-bibr">47</a>,<a href="#B48-polysaccharides-05-00024" class="html-bibr">48</a>,<a href="#B49-polysaccharides-05-00024" class="html-bibr">49</a>,<a href="#B50-polysaccharides-05-00024" class="html-bibr">50</a>,<a href="#B51-polysaccharides-05-00024" class="html-bibr">51</a>,<a href="#B52-polysaccharides-05-00024" class="html-bibr">52</a>,<a href="#B53-polysaccharides-05-00024" class="html-bibr">53</a>,<a href="#B54-polysaccharides-05-00024" class="html-bibr">54</a>,<a href="#B55-polysaccharides-05-00024" class="html-bibr">55</a>,<a href="#B56-polysaccharides-05-00024" class="html-bibr">56</a>,<a href="#B57-polysaccharides-05-00024" class="html-bibr">57</a>,<a href="#B58-polysaccharides-05-00024" class="html-bibr">58</a>].</p> "> Figure 10
<p>Agarose native gel electrophoresis of various macromolecules. Electrophoresis of five different model proteins was performed using His/MES buffer at pH 6.1 (<b>A</b>) and Tris/Gly buffer at pH 8.3 (<b>B</b>). Additionally, electrophoresis of five high-molecular-weight proteins was conducted using His/MES buffer (<b>C</b>). These data were assembled and reorganized from ref. [<a href="#B60-polysaccharides-05-00024" class="html-bibr">60</a>] and unpublished data.</p> "> Figure 11
<p>Zymography of agarose native gel electrophoresis. (<b>A</b>) Schematic illustration of zymography. (<b>B</b>) ß-Galactosidase stained by the substrate, X-gal. C. ß-Lactamase stained by the substrate, nitrocefin. These data were assembled and reorganized from ref. [<a href="#B64-polysaccharides-05-00024" class="html-bibr">64</a>] (<b>B</b>) and unpublished data (<b>C</b>).</p> "> Figure 12
<p>Expression and purity analysis of HEK293 cell culture supernatant expressing mAb. One percent flat agarose gels at 100 V for 1 hr for protein analysis and for 30 min for DNA analysis: 7 μL of supernatant per lane and the cell culture media harvested at day 0 (d0) and day 7 (d7). Panel (<b>A</b>): CBB staining. The mAb and HCPs are indicated by arrow and bracket. Panel (<b>B</b>): gel stained with fluorescent GelGreen dye. DNA staining is indicated by arrow and bracket. Panel (<b>C</b>): blotted membrane stained with Colloidal Gold Total Protein Stain. Panel (<b>D</b>): immuno-stained blot for HCPs with anti-HEK293 cell antibody. These data were assembled and reorganized from ref. [<a href="#B64-polysaccharides-05-00024" class="html-bibr">64</a>], where details are given.</p> "> Figure 13
<p>Gel extraction of proteins. (<b>A</b>) Schematic illustration of gel extraction. (<b>B</b>) Purification of monoclonal antibodies using gel extraction method. Rabbit monoclonal antibodies were expressed and secreted in the medium using HEK293 cells. These antibodies were then purified using the gel extraction method and analyzed by agarose native gel electrophoresis. The negative control for this data is in the CM lane of <a href="#polysaccharides-05-00024-f012" class="html-fig">Figure 12</a>. Unpublished data are presented. This is an original diagram drawn by us.</p> "> Figure 14
<p>Western blotting. (<b>A</b>) Schematic illustration of Western blotting. (<b>B</b>) HEK293 membrane fraction expressing human or mouse PLXDC2. (<b>C</b>) Recombinant S-CoV-2 alpha, delta, omicron BA1.1.529 or omicron BA2 spike protein (1 μg/lane) was loaded. The blot was stained by a neutralizing humanized mAb (Abwiz Bio Inc., San Diego, CA, USA, #G10 × A1) (1:5000) against S-CoV-2 spike protein. Left: agarose native gel electrophoresis. Agarose native gel electrophoresis was done at 100 V for 90 min. (<b>a</b>) CBB staining of agarose gel. (<b>b</b>) Detected antibody on contact blotting with 20 s exposure. Right: SDS-PAGE. SDS-PAGE (10–20% gradient gel, reducing condition) was run at 300 V for 35 min. (<b>c</b>) CBB staining of SDS-PAGE. (<b>d</b>) Detected by antibody on electroblotting with 20 s exposure. Details of this method are described in refs. [<a href="#B71-polysaccharides-05-00024" class="html-bibr">71</a>,<a href="#B73-polysaccharides-05-00024" class="html-bibr">73</a>]. Unpublished data are presented.</p> "> Figure 14 Cont.
<p>Western blotting. (<b>A</b>) Schematic illustration of Western blotting. (<b>B</b>) HEK293 membrane fraction expressing human or mouse PLXDC2. (<b>C</b>) Recombinant S-CoV-2 alpha, delta, omicron BA1.1.529 or omicron BA2 spike protein (1 μg/lane) was loaded. The blot was stained by a neutralizing humanized mAb (Abwiz Bio Inc., San Diego, CA, USA, #G10 × A1) (1:5000) against S-CoV-2 spike protein. Left: agarose native gel electrophoresis. Agarose native gel electrophoresis was done at 100 V for 90 min. (<b>a</b>) CBB staining of agarose gel. (<b>b</b>) Detected antibody on contact blotting with 20 s exposure. Right: SDS-PAGE. SDS-PAGE (10–20% gradient gel, reducing condition) was run at 300 V for 35 min. (<b>c</b>) CBB staining of SDS-PAGE. (<b>d</b>) Detected by antibody on electroblotting with 20 s exposure. Details of this method are described in refs. [<a href="#B71-polysaccharides-05-00024" class="html-bibr">71</a>,<a href="#B73-polysaccharides-05-00024" class="html-bibr">73</a>]. Unpublished data are presented.</p> ">
Abstract
:1. Introduction
2. Mechanism of Molecular Sieving
3. Polysaccharide vs. Polyacrylamide
4. Agarose Gel vs. Protein Gel
5. Denaturing vs. Native Gel Electrophoresis
6. Effect of Small Pore Structure
7. Application of Agarose Gel Electrophoresis
7.1. Nucleic Acids
7.1.1. Gel Shift Assay
7.1.2. Comet Assay
7.2. Proteins
7.2.1. Native Gel Electrophoresis
7.2.2. Activity Staining
7.2.3. Detection of Contamination
7.2.4. Purification of Protein by Gel Extraction
7.2.5. Native Western Blotting by Contact
8. Conclusions
Funding
Acknowledgments
Conflicts of Interest
References
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Arakawa, T.; Nakagawa, M.; Sakuma, C.; Tomioka, Y.; Kurosawa, Y.; Akuta, T. Polysaccharide as a Separation Medium for Gel Electrophoresis. Polysaccharides 2024, 5, 380-398. https://doi.org/10.3390/polysaccharides5030024
Arakawa T, Nakagawa M, Sakuma C, Tomioka Y, Kurosawa Y, Akuta T. Polysaccharide as a Separation Medium for Gel Electrophoresis. Polysaccharides. 2024; 5(3):380-398. https://doi.org/10.3390/polysaccharides5030024
Chicago/Turabian StyleArakawa, Tsutomu, Masataka Nakagawa, Chiaki Sakuma, Yui Tomioka, Yasunori Kurosawa, and Teruo Akuta. 2024. "Polysaccharide as a Separation Medium for Gel Electrophoresis" Polysaccharides 5, no. 3: 380-398. https://doi.org/10.3390/polysaccharides5030024