A good question. Please read this article at below link:
"Why aren’t SDS-PAGE gels horizontal?"
http://bitesizebio.com/10699/why-arent-sds-page-gels-horizontal/
A good question. Please read this article at below link:
"Why aren’t SDS-PAGE gels horizontal?"
http://bitesizebio.com/10699/why-arent-sds-page-gels-horizontal/
Interesting question, there are several reasons but the main ones are these:
- Gels: Correct preparation of stacking/separation gels can not be done horizontally (but this is not a problem with premade gels ... except that fact that the wells would be in the wrong place!).
- Thickness: The polyacrylamide gels are so thin compared to agarose gels that you would not be able to make wells large enough for your sample. ;)
- Gravity: Vertical position ensures that most (if not all) of your sample will enter the gel.
- Design: By now, Western Systems are pretty standardized and vertical setups work best.
- Buffers: In some PAGE setups, you want different buffers inside and outside the gel chamber. That would be quite the challenge in a horizontal system!
Yan-Yeu Tau: Those are all good points, but I rarely meet anyone casting their own gels these days. :)
@ Nadia,
I agree with you, more researchers are using precast SDS-PAGE gels these days because it is convenient and easy to use. I use them too. However, I believe that, in some regions, students/researchers have to case their own gels (and re-use combs, glass plates, sapcers....) just to save money due to restrained fundings.
@ Ranjan,
Using a 'horizontal' system to run SDS-PAGE is possible. See the attached paper.....But, I think that most people go by vertical system.
owing to that proteins and small DNA fragments are too small
Polyacryamide is more packed
intermingling of R groups of amino acids
There are also horizontal systems for proteins on the market. With those you have to use precast gels which is more expensive than the easely made self casted vertical gels. They do privide some very nice features though. Horizontal gels are much thinner than vertical ones (0,5 - 0,65 mm) and therefore provide a much better resolution especially with proteins of low molecular weight. The gels run nearly without buffer and lay on an evenly cooled ceramic plate. You can also load much higher sample numbers (25-52). So it's up to you and the equipment your lab provides.
@ Kristin,
Do you have the info of the companies selling that kind of precast gels you described. I am interested in taking a look. Thanks.
Frank
We cant seperate the proteins in a horizontal agarose gels since they having higher molecular size compared with DNA .Proteins are amphoteric in nature but DNA are negatively charged molecules.PAGE (Polyacrylamide Gel Electrophoresis), is an analytical method used to separate components of a protein mixture based on their size. The technique is based upon the principle that a charged molecule will migrate in an electric field towards an electrode with opposite sign.The general electrophoresis techniques cannot be used to determine the molecular weight of biological molecules because the mobility of a substance in the gel depends on both charge and size. To overcome this, the biological samples needs to be treated so that they acquire uniform charge, then the electrophoretic mobility depends primarily on size. For this different protein molecules with different shapes and sizes, needs to be denatured(done with the aid of SDS) so that the proteins lost their secondary, tertiary or quaternary structure .The proteins being covered by SDS are negatively charged and when loaded onto a gel and placed in an electric field, it will migrate towards the anode (positively charged electrode) are separated by a molecular sieving effect based on size. After the visualization by a staining (protein-specific) technique, the size of a protein can be calculated by comparing its migration distance with that of a known molecular weight ladder(marker).
Hi Ranjan, yours is an old question, but maybe you are still interested...
Keep in mind the physical/chemical nature of the two types of gels:
- agarose gels are just a condensed medium, a sugary solution that is solid at RT. It's simple and fast to pour it in a tray and let it dry;
- polyacrylamide is obtained through a polymerization reaction which needs to occur in the absence of oxygen, so you need a closed environment to obtain it, simple enough between two glass slabs.
For the rest, horizontal or vertical makes no difference, the molecules move because of an electric field, not because of gravity that in such dense media would be neglectable. In fact isoelectric focusing of proteins is done horizontally now (on industrially pre-cast strips), but was done vertically previously in home-made capillaries, simply because of technical easiness of preparation. Same for PAGE gels: as Kristin was mentioning, there is expensive equipment that allows running PAGE horizontally on thin strips for high throughput, sensitivity, and reproducibility purposes, but with self-made gels it's easier to do it in vertical glass slabs.
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