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SDS-PAGE help please

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SDS-PAGE help please

Postby carmenh » Fri Feb 27, 2009 10:31 pm

So SDS is used in Polyacrylamide gel electrophoresis, but I'm still having a little difficulty understanding what it does.

As far as I can tell, its a detergent that not only breaks up a protein into polypeptides and gives an overall negative charge to the polypeptides. If I remember correctly, gel electrophoresis is used to sort out MW of DNA fragments, which naturally carry a negative charge. Amino acids, on the other hand, can have negative, positive, or neutral charges. So why must we give everything the same overall negative charge? If the numerical charge is the same, won't it effect the results of the gel so that we can't interpret the results?

For an upcoming test in cell biochemistry, we must understand electrophoresis and how the addition or lack of SDS will affect the electrophoresis; I'm assuming this is for proteins. Any insight into my dilemma would be greatly appreciated.
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Re: SDS-PAGE help please

Postby Sepals » Wed Apr 01, 2009 3:49 pm

So these move towards a potive pole:

http://www.bio.davidson.edu/COURSES/GEN ... SPAGE.html
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Re: SDS-PAGE help please

Postby JackBean » Mon Sep 14, 2009 8:22 pm

Hi,
first of all, SDS does NOT break proteins into smaller peptides.
As you wrote, aminoacids can have negative/positive charge, so if you put it into electric field, it will move to electrode with the other charge. For this reason you add SDS, which binds to all proteins in approx. the same ratio and gives unique charge/molecular mass, so you are dividing only in acordance to molecular mass. If you did not add SDS, than also the overall shape would influence the movement, that is "native electroforesis" ;)

Sepals wrote:So these move towards a potive pole:

http://www.bio.davidson.edu/COURSES/GEN ... SPAGE.html


Well, that is page, from which I have learned about SDS-PAGE several years ago :) And I remeber it still quite well :)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby Mercury33 » Tue Sep 29, 2009 9:01 pm

I've just read the above and would like a more detailed account of the SDS - I didn't realise you could run a gel without the SDS? I understand that it gives a negative charge to the proteins traveling toward the positive pole - sorry, you couldn't explain why you sometimes don't have SDS? Just find it interesting :)
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Postby canalon » Tue Sep 29, 2009 9:04 pm

Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
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Re: SDS-PAGE help please

Postby MJD » Thu Oct 01, 2009 2:58 am

Okay. So it seems that there is a little confusion about what we are taking about. Electrophoresis is a process used for the separation of cellular proteins (polyacrydimide) and DNA (agarose gels). SDS-PAGE is electrophoresis for proteins that utilizes SDS and denaturing conditions to linearize the proteins, which will separate out by size. SDS binds to proteins in specific ratios of SDS to protein molecules. The denaturing conditions typically involve agents that have sulfur or cysteine groups capable of competiting for and breaking disulfide bonds that hold the protein together in its native conformation (highly folded). DTT and B-mercaptoethanol are typical reducing agents used for this purpose. Once the proteins are linearized by the DTT/B-mercaptoethanol, then the SDS binds to the proteins preventing them from refolding. Typically, the samples are boiled to enhance the reduction and linearization of the proteins prior to loading them on the gel. The gel consists of combination of reagents that can be found here : https://webspace.utexas.edu/mjd57/www/M ... 20Cell.PDF

A combination of polyacrylamide, deionized water (ddH20), Tris Buffer (to maintain pH), Ammonium Persulfate (APS), SDS, and TEMED are mixed together to create a polyacrylamide gel that can be thought as a matrix net like structure. The resolving gel is where the protein separate (resolve), and the stacking gel is overlayed second on the top (where the samples are eventually loaded). The stacking gel is a lower percentage polyacrylamide (meaning that it has bigger holes and the protein move through very easily). The comb is insert into the stacking gel while it is still liquid creating wells, where the samples are loaded. TEMED catalyzes the polymerization of the acrylamide polymers by enhancing the free radical production of APS, which results in the gel matrix. The size of the pores in the gel is inversely proportional to the percentage of the gel (amount of acrylamide used). The gel is placed in a running gel box with a certain amount of running buffer (specially formulated to carry charge). The current runs from the top to the bottom of the box pulling the protein from the top of gel towards the bottom. Proteins migrate based on size with the small proteins migrating the fastest and the largest protein migrate slowest. A blue dye front is sometimes used (put into the sample buffer with SDS/reducing agents) to identicate when the proteins have reached the bottom of the gel. Also, a colored protein marker standard (proteins with a known characterized molecular weight) are loaded to guide electrophoresis progression. Once the gel is finished running (either the blue dye runs off), you stop it one marker below your protein size of interest (say the protein is 42 kilodaltons and there are markers for 250, 150, 100, 75, 50, 37, 25, 20 and 15. Then, you want to have at least the 37 kDa band still visible on the bottom of the gel. Typically, a gel is run for 1-1.5hr at 100-150V, it just depends on the person and the lab I might add.

Gel Transfer to Membrane for Western (immunoblot)

Next, the gel is remove from the glass plates and it is set up in a cassette that consists of a sandwich that contains filter paper and a nitrocellulose or PDVF membrane. The cassette is placed in an apparatus similar to the running gel box, but this time the transfer buffer used contains methanol, which facilitates the migration of the protein from the gel matrix onto the membrane. The cassette in placed in such a way that the current runs through the gel matrix towards the membrane. Usually this process generates considerable heat and is therefore put in a 4 degree coldroom, beer cooler or set in ice. It runs for about 1hr at 100V. When it is finished, the proteins have been smacked onto the membrane and the colored molecular weight markers are clearly visible.

So What's the Point? You can detect the amount of protein variation between different cell lines, cell types or treatments. Antibodies to the specific proteins (if they are highly specific) generate a single band at the expected molecular weight. So you can see how your treatment changes a particular protein over time and at different concentrations, etc.

Here is another good website from Fisher Scientific:
http://www.piercenet.com/Proteomics/bro ... 36EC5B641E


DNA agarose gel;

Similar idea here, but different buffers, apparatus and gel. The gel is made from agarose and instead of using antibodies to detect protein, you are using Ethidium Bromide (EtBr), an agent that intercalates into the DNA. When hit with UV light, the EtBr gives off a characteristic fluorescence (glow) that allows for visual confirmation of your results and enable one to take a picture with a gel documentation camera.

If you want more information about either of these processes. Go to one of the websites above or alternatively go to http://www.ncbi.nlm.nih.gov/sites/entrez
and type in SDS-page or agarose gel

I hope that makes more sense now. A virtual lab for DNA agarose electrophoresis is available here:
http://www.classzone.com/cz/books/bio_0 ... lLabs.html
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Postby JackBean » Thu Oct 01, 2009 11:45 am

1) the elfo is not restricted only PAGE - proteins/agarose - DNA
2) you definitelly don't have to use antibodies to detect proteins (how could you than stain e.g. crude extracts or unknown proteins?)
3) you forgot to writte, that DNA has negative charge by itself (the sugar-phosphate backbone), so it doesn't need anything like SDS
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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