Gel Electrophoresis! Anode/Cathode??

[axp107]

Well, I know gel electrophoresis works like an electrolytic cell, meaning the anode is +ive and cathode is -ive.

1) So which direction do things flow in gel electrophoresis? Does it depend on charge? (I know it depends on weight also but let's say we're talking about charge only here)

Anything else thats important to know about gel electrophoresis?

2) Isoelectric points! These confuse the hell out of me.

Consider an amino acid... its the point where the OH group is -ive and the NH3 group is +ive right

What does having an acidic or basic isoelectric point mean??

Also when it comes to gel electrophoresis.. how do isoelectric points come into play?

For example:

If a solution of amino acids at a pH of 8 underwent electrophoresis, which of the following would move furthest towawrd the anode?

The answer is: The amino acid with an acidic isoelectric point will be negatively charged at a pH of 8. Is it b/c below 7, itll be neutral.. but once the pH increases, it becomes negative. But why does being more negative mean it'll travel the farthest?

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[ahmedqman]

In gel electrophoresis for DNA, DNA is negatively charged so it always moves towards positive. the anode is obviously positive then. the same works with amino acid separation if I recall.

Now, the isoelectric point of an amino acid is the pH where it's zwitterion predominantly exists. The isoelectric point depends on the side chain of an amino acid if I recall.

Now if you have a side chain that makes the isoelectric point acidic, at higher pHs, everything becomes deprotonated and it will become more negative.

I can look in my orgo textbook for you if you need examples of amino acids with acidic isoelectric points vs. basic isoelectric points.

To simply answer your final question: Because gel electrophoresis of amino acids is based on differing degrees of attraction to the positive side of the gel chamber

 

[themule]

Well, I know gel electrophoresis works like an electrolytic cell, meaning the anode is +ive and cathode is -ive.

1) So which direction do things flow in gel electrophoresis? Does it depend on charge? (I know it depends on weight also but let's say we're talking about charge only here)

Nucleic acid GE has the DNA flowing to the anode negative PO4 groups moving to positive terminal - incidentally I always remember this as "run to red". The gel in GE acts as a matrix that separates NA by size not charge. A pH gradient gel can separate AA by charge due to the isoelectric point of AA. When the AA reaches it's isoelectric point it will have NH3+ and COO- and is therefor not carrying a charge.

Anything else thats important to know about gel electrophoresis?

2) Isoelectric points! These confuse the hell out of me.

Consider an amino acid... its the point where the OH group is -ive and the NH3 group is +ive right

What does having an acidic or basic isoelectric point mean??

Also when it comes to gel electrophoresis.. how do isoelectric points come into play?

For example:

If a solution of amino acids at a pH of 8 underwent electrophoresis, which of the following would move furthest towawrd the anode?

The answer is: The amino acid with an acidic isoelectric point will be negatively charged at a pH of 8. Is it b/c below 7, itll be neutral.. but once the pH increases, it becomes negative. But why does being more negative mean it'll travel the farthest?

So the isoelectric point of AA is the pH where the AA exists as a zwitterion. An AA exists as a positive ion at a pH less than the pKa where it will have NH3+ and COOH above the pKa of the Carboxyl group pH>3 it exists as a zwitterion until it reaches the pKb of the amine group ph>9 when the NH3+ deprotonates and exists as NH2. So the AA goes from positive at low pH to negative at high pH. Hope this helps.

TM

 

[Kaydubz]

In a galvanic cell, the anode is (-), and the focus is on pushing away electrons from the anode to supply power. In an electrolytic cell and gel electrophoresis, the anode is (+) and the focus is on attracting the negative ions in solution from inputted power.

So, the more negatively charged the protein, the more it will move towards the (+) anode.

The isoelectric point is simply the pH where all the charges in a protein balance out to 0. With no ionizable side chains, it's the average of the pKa's of the carboxylic acid and amine.

To make it simple, just relate the pH of solution to the pI. If the pH is below the pI, the solution is acidic to the amino acid and there are a lot of hydrogens hanging around that want to protonate the amino acid and make it positively charged. If the pH is above the pI, there are a lot of hydroxide ions that want to deprotonate the amino acid and make it negatively charged.

In your problem, you have an acidic amino acid, but that doesn't mean it'll be neutral below a pH of 7. It will be neutral at a pH equal to its isoelectric point. Any pH above that (like 8) will deprotonate it and send it toward the (+) anode.

 

[axp107]

Thanks for confirming guys.

Is electrophoresis ever performed to separate positively charged compounds?
where anode is at top and cathode is at bottom

 

[axp107]

Also would we have to know how to read electrophoresis gels for the MCAT... with all the bands etc.. I have no idea how.

 

[themule]

Also would we have to know how to read electrophoresis gels for the MCAT... with all the bands etc.. I have no idea how.

I highly doubt this. I've never seen them on any of the practice exams. There is really nothing to reading them. Primarily what you are looking for is size differentiation. So anytime you run a gel you also run a size or mass ladder so that you can tell how big the band patterns are. So really pretty easy. But I wouldn't worry about reading a gel but when would you use it and how would you use it would be what I would know.

TM

 

[greg1184]

To read a gel, the smaller molecules migrate farther than the larger molecules. To determine size, you have to run a standard beside your sample like HindIII. However this is way beyond the scope of the MCAT. In the lab I work at we do gels all the time. This is relevant to DNA molecules.

 

[ssquared]

2) Isoelectric points! These confuse the hell out of me.

Consider an amino acid... its the point where the OH group is -ive and the NH3 group is +ive right

What does having an acidic or basic isoelectric point mean??

Also when it comes to gel electrophoresis.. how do isoelectric points come into play?

For example:

If a solution of amino acids at a pH of 8 underwent electrophoresis, which of the following would move furthest towawrd the anode?

The answer is: The amino acid with an acidic isoelectric point will be negatively charged at a pH of 8. Is it b/c below 7, itll be neutral.. but once the pH increases, it becomes negative. But why does being more negative mean it'll travel the farthest?

I think I know what's confusing you: Kaplan teaches zwitterions in the context of a pH gel. The idea is that the gel has a gradient going from low to high pH; an AA will move to the pH equal to its pI. At that point, it is effectively neutral, and will not move through the gel.

If the AA is more negative, it's going to be increasinly attracted to that hunk of positive charge at the end of the gel. The more negative, the further it will go. The same principle works for a gel separating based on size; a smaller protein will go further than a larger one.

As for the acidic/basic isoelectic point, it is what it sounds like: the pI of the AA is either below or above a pH of 7. So, for the dissociation, if it's positive below the pI, it will be negatively charged above it.

 

[tennisboy85]

In a galvanic cell, the anode is (-), and the focus is on pushing away electrons from the anode to supply power. In an electrolytic cell and gel electrophoresis, the anode is (+) and the focus is on attracting the negative ions in solution from inputted power.

Is that right? I didn't know that anode is (+) in an electrolytic cell, I thought it was still (-) because it's still the site of oxidation. But the difference is you're running a current and forcing the electrons to go the other way?

 

[Kaydubz]

Is that right? I didn't know that anode is (+) in an electrolytic cell, I thought it was still (-) because it's still the site of oxidation. But the difference is you're running a current and forcing the electrons to go the other way?

The electrons are still going the same way. Oxidation still happens in the anode and electrons are propelled along the wire away from the anode.

If you think about a galvanic cell, what is happening? A spontaneous reaction oxidizes the metal, sending electrons away and putting positive cations (the oxidized metal) into solution. The anode is labelled (-) because the electrons are spontaneously pushed away. But positive cations are also being spontaneously pushed away! So why wouldn't the anode be (+) to push away the cations? Simply because the purpose of the cell is to force electrons away generating power, so we use the relation between (-) anode and (-) electrons.

In the electrolytic cell, oxidation still takes place at the anode, but is forced to by a power source. Electrons are still being sucked away from the metal at the anode, and cations are still being put into solution as oxidation takes place. So again, if could be (-) or (+) depending how you look at it. But since in the electrolytic cell the focus is on the ions in solution (as in gel electrophoresis), the anode is labeled (+) to push away (+) cations and attract anions.

And don't forget that 'current' is opposite the electron flow too.

 

[tennisboy85]

And don't forget that 'current' is opposite the electron flow too.

Ahahaha, you caught me there. Thanks.

 

[nontradnontrad]

I've got a Kaplan question (Full Length 2, C/P #9) on this subject where the answer seems backward.

When placed in an electric field:

it says that the molecule in which the positively charged amino groups would outnumber the negatively charged carboxylate groups will migrate toward the cathode at pH 7.0. I chose the molecule that would be negatively charged at pH 7 because it (-) would migrate to a (positive) cathode.

Now, I know it would go where the positive ions go in isoelectric focusing, but that seems like it would be because it migrates toward an anode. Welcoming any clarification on this...

 

[Zone05]

Good info! Thanks for the clarification I had this issue too 🙂

 

[sunshine859]

I've got a Kaplan question (Full Length 2, C/P #9) on this subject where the answer seems backward.

When placed in an electric field:

it says that the molecule in which the positively charged amino groups would outnumber the negatively charged carboxylate groups will migrate toward the cathode at pH 7.0. I chose the molecule that would be negatively charged at pH 7 because it (-) would migrate to a (positive) cathode.

Now, I know it would go where the positive ions go in isoelectric focusing, but that seems like it would be because it migrates toward an anode. Welcoming any clarification on this...

What did Kaplan say was the correct answer on this one?

 

[PlsLetMeIn21]

According to TBR, in electrical fields such as electrophoresis gels, cations migrate to the cathode and anions migrate to the anode. So for a protein with more (+)-charges than (-)-charges, it would have an overall positive charge and migrate to the cathode.

Electrophoresis uses a charged capacitor to create the electric field. In the circuit surrounding the gel, the cathode of the battery is connected to the anode of the capacitor and the anode of the battery is connected to the cathode of the capacitor. The capacitor starts neutral and over time the battery's cathode pushes (+)s to the capacitor's anode, giving it an overall (+) charge on the plate. The battery's anode pulls (+)s from the capacitor's cathode, leaving a (-) charge on the plate. The capacitor surrounding the gel has a (+) anode and a (-) cathode, which is why anions migrate through the gel to the anode and cations migrate through the gel to the cathode.

TBR orgo books explain this super well, so if you can't understand what I'm saying then read their description.