I know that DNA molecules or proteins can be separated in electrophoresis because the electrical charge is used to pull the DNA through the gel. So instead of using electrical, can I using physical impact like pumping ?
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$\begingroup$ Do you have a particular reason for wanting to use pressure instead of electricity? Any specific application in mind? $\endgroup$– fileunderwaterCommented Jul 1, 2013 at 13:40
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$\begingroup$ Well ... A lot of reason why i think pressure is better than electricity. Pressure will work with any molecule even they don't have charged, so it give better result without denaturing the protein. It's also safe budget, safe time when protein electrophoresis need to using chemical substrate for denaturing the protein. Pressure also safety for a diy bio like me when i don't want to working with high electricity... And i think with pressure i have more and more application, may be a machine that can purify protein, detect viruses in real time from good protein separate result... $\endgroup$– DucFabulousCommented Jul 1, 2013 at 15:18
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$\begingroup$ Much of this comment should be included in your question. It gives background and makes the question easier to answer, and also shows what particular problems you have been thinking and that you are looking for DIY solutions. $\endgroup$– fileunderwaterCommented Jul 1, 2013 at 17:49
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3 Answers
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If you tried to set up a system in which movement of DNA molecules through a gel matrix was driven by pumping buffer, the gel would become compressed. I am old enough to remember running vertical agarose gel electrophoresis overnight, and these gels would collapse under normal pressure to about 2/3rds of their initial height. A gel porous enough to give useful separation of DNA molecules just isn't strong enough to withstand pumping.
answered Jun 29, 2013 at 17:57
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not from gels but physical force can be used to separate molecules.. e.g. density centrifugation, filtration. Closest to what you are asking is size exclusion chromatography in HPLC where molecules are separated based on size and high pressure is maintained in order to pump the solution (containing the analytes) through the stationary phase.
As Alan Boyd mentioned, the gels cannot withstand high pressure. In HPLC silica beads are used (sometimes coated with specific resins for e.g. in ion-exchange chromatography).
The principal differences between electrophoresis and HPLC (chromatogrphy) are:
- HPLC relies on differential interaction of the molecules with the stationary phase (i.e beads) whereas electrophoresis is purely based on the hydrodynamic size of the molecules.
- HPLC uses pressure to keep the entire mobile phase (solution) pumping through the stationary phase, whereas electrophoresis uses electric field to move the molecules.
- HPLC can also be used for uncharged molecules
Size exclusion and gel-filtration chromatographies resolve the molecules based on size as in the case of electrophoresis but as I previously mentioned, the molecules need not be charged. The techniques used for analysis of molecules in both techniques can be similar. For e.g UV absorption/fluorescence. An advantage of HPLC is that it can be connected to other analyzers like Mass Spectrometer, Flame Photometer etc.
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$\begingroup$ Hello, thank you so much. What is the gel HPLC using for separate molecules ? What different between HPLC and Electrophoresis (i mean in the result) $\endgroup$ Commented Jul 1, 2013 at 1:51
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$\begingroup$ Hello, so if i replace the gel using in electrophoresis with the gel-filtration using in HPLC. That mean i can use pressure to separate molecules instead of electrical ? $\endgroup$ Commented Jul 1, 2013 at 2:29
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$\begingroup$ yes. you can. but HPLC is a different setup altogether. there are pre-packed columns available, which contain the beads. $\endgroup$– WYSIWYGCommented Jul 1, 2013 at 2:31
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$\begingroup$ Amazing ! As what i think in my dream, there are a method like that. Thank you so much ! $\endgroup$ Commented Jul 1, 2013 at 2:36
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No, you can't use mechanical pumping for gel electrophoresis. The nature of electrophoresis is the create a uniform electric field through the gel, and this electric field stimulates motion from the charged DNA/protein.