3. Polyacrylamide gel electrophoresis (PAGE) can be used to separate proteins by

Question

3. Polyacrylamide gel electrophoresis (PAGE) can be used to separate proteins by size. First you have to coat the proteins with a special detergent, SDS, which has a negative charge. Then apply the protein sample to the top of the gel, which has pores in it beg enough for proteins to travel through. The you apply and electric current across the gel...the proteins will migrate through the gel towards which electrode? (What charge do they have on them?) Why do you have to coat the proteins with negatively charged detergent? (What charge would a protein have in its "native conformation"?.. this is a bit of a trick questions..think about it.. Small proteins will migrate through the gel faster than larger ones. By comparing the mobility of your sample to a set of standard proteins of known molecular weight, you can determine the molecular weight of the proteins in your sample. dharged dete gent

Explanation / Answer

I'm starting from the last part of your problem. That is about the charge in its native state. You know that protein is made up of amino acids. So each amino acid has amine and carboxyl functional group. In isoelectric point (pI), the amine group is present as NH3+ & carboxyl as COO-. So one plus and one minus charge is present. And the net charge is zero i.e., neutral. In this state, it is called as zwitterion. The charge totally depends on the pH of the medium and on that pI depends. In higher pH, the ions stay as NH2 & COO-. So it becomes negative. In lower pH the ions stay as NH3 & COOH. So it becomes positive. This higher and lower pH are with respect to the isoelectric point ( a pH where the charge is neutral). The charge of the protein depends on the charge of amino acids. In native conformation, the protein stays at its pI. So it has neutral charge.

Moving on to the previous part. SDS is a strong denaturing agent which has a negative charge. The modus operandi is like this - SDS helps to break the bonds which are used to keep the peptide chain tighty folded. If the bonds are not destroyed, the protein would not have moved through the tiny pores of that gel. As SDS is negatively charged when it binds to the polypeptide backbone, it imparts uniform negative charge to the linear peptide chain. Now, protein along with SDS can easily move through the minute gel pores. SDS is the standard molecule for PAGE as it is the most efficient molecule among all.

Now remember one thing, this method is applied when we need to seperate protein on the basis of their size not charge. As I've mentioned earlier that it imparts uniform charge. So, when the parameter charge is uniform, we can seperate it on the basis of another parameter that is size.

Now, it has a simple anode - cathode system. As per the principle of electrophoresis, a charged molecule travels through a medium because it is attracted to the opposite charge. You know that positive and negative attraction. Now as the protein is negatively charged (anion) , it must be attracted by the positive electrode. The name of the positive electrode is anode. Because anion goes toward the anode.

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