Electrophoretic Separation & Zwitterion

[weanprednisone]

Electrophoretic separation at pH 6 of a sample of polypeptide 1 (mw 1000) polypeptide 2 (mw 500) and polypeptide 3 (mw 100) would result in which of the following?
(Note: the isoelectric point of each polypeptide occurs at pH 6)
Isoelectric point table
Polypeptide 1 pI=6
Polypeptide 2 pI=2
Polypeptide 3 pI=9

A. P1 will move the farthest
B. P2 will move the farthest
C. P3 will move the farthest
D. None will move.

My thoughts:
to electrophoretically separate, we need a neg. charge and light mw polypeptide.
P1 is neutral and super heavy, so it will not move much. Therefore A is false.
P2 will be negative at pH6, but is heavier than P3
P3 will be positive at pH6, but is lighter than P2
We are left with B and C. Which one overrides the other? size or charge?

---

Members don't see this ad.

 

[theonlytycrane]

The negative charge is usually at the end with the wells so I think that p2 would be the one moving toward the positive end as p3 is still positive at pH = 6

 

[aldol16]

We are left with B and C. Which one overrides the other? size or charge?

Think about how you're designing this. In electrophoresis, what kind of charge moves where? Think about how DNA separation works.

 

[BeMD13]

Isn't it that none will move bc the ph and isoelectric points are the same?

 

[aldol16]

But you give an isoelectric point table showing that the isoelectric points are all different...

 

[BeMD13]

But you give an isoelectric point table showing that the isoelectric points are all different...

But it also says they reach those points at the ph that they are in so they would all be zwitterions with no charges. I'm pretty sure I did this exact question and that was the answer and explanation.

 

[aldol16]

But it also says they reach those points at the ph that they are in so they would all be zwitterions with no charges. I'm pretty sure I did this exact question and that was the answer and explanation.

I'm not sure what you're saying here. Care to elaborate?

 

[weanprednisone]

@aldol16 and @theonlytycrane
Maybe the question didn't translate well over... Here is my question again

@BeMD13 It is a KA question, but I manipulated it a little.

Thanks in advance.

 

[weanprednisone]

Or here is another way of asking this question:
P1 is negative; MW=5000
P2 is positive; MW=200
Which one will go further?
I am guessing P1 will go further, despite the MW.

 

[aldol16]

Okay, I'm no molecular biologist, so a molecular biologist, feel free to correct me. The basis of electrophoresis is using an electric field to separate particles. Particles will move through a gel in that electric field based on their charge and size. In other words, the electric field is providing the impetus (force) for their movement. If they're uncharged, would they migrate towards a positive charge? If they're positively charged, would they migrate towards a positive charge?

I guess a better way to put it would be that particles move based on their size, within the parameters given by their charge. Kind of like having a huge ball of charge on one side of the pool. Would something without charge move across the pool? How about something with the same charge as the ball? Opposite charge? Now, what if you had two things with opposite charges relative to the ball of charge. If they move, what other factor will govern how fast they move through the pool, a viscous substance?

 

[BeMD13]

@aldol16 and @theonlytycrane
View attachment 200438
@BeMD13 It is a KA question, but I manipulated it a little.

Thanks in advance.

Here is the original Khan Academy question with their explanation. Honestly, I'm still confused by this so if anyone else can elaborate, please do.

 

[weanprednisone]

Ok, so like we have all been saying. A negative and light weight polypeptide will move the farthest, because the + electrode is at the end, and light weight things can move faster than heavy things.
Just like ion-exchange chromatography, is we had positive resins, then positive proteins will elute our first, then neutral, then negative resins will barely elute or elute last, because it has a strong ion-ion attraction.

Based on the original KA question, we can use the reasoning that the polypeptides' pI=6, then all the proteins in pH=6 will be neutral charge.

Therefore, neutral charged polypeptides' will not move at all. (So my diagram was incorrect regarding the P1)

But this question asks if we have a
Polypeptide with a negative charge with heavy MW
Vs.
Polypeptide with a positive charge with light MW

I think the answer would be P2 regardless of the size, because the + in the gel will attract the -. But even though P3 is light weight (swim faster) it has no attraction to go towards the - side, it would actually go the opposite way (towards the -side)...

Is that a valid answer/reasoning?

 

[weanprednisone]

Here is the original Khan Academy question with their explanation. Honestly, I'm still confused by this so if anyone else can elaborate, please do. View attachment 200439

Lmk if you still don't get the KA question, BC I am able to understand where they got that answer.

 

[aldol16]

Based on the original KA question, we can use the reasoning that the polypeptides' pI=6, then all the proteins in pH=6 will be neutral charge.

This is not compatible with the original question you posted above. You gave a table of values for the pI's of the polypeptides, all of which were different.

 

[weanprednisone]

This is not compatible with the original question you posted above. You gave a table of values for the pI's of the polypeptides, all of which were different.

This is confusing I understand. But I wrote this question based on a KA question, which is posted by BeMd. My question and the original is different.

 

[Phoodie]

This is confusing I understand. But I wrote this question based on a KA question, which is posted by BeMd. My question and the original is different.

Did you write the problem in your original post yourself? You have contradictory info which seems to be confusing everyone including yourself:

Electrophoretic separation at pH 6 of a sample of polypeptide 1 (mw 1000) polypeptide 2 (mw 500) and polypeptide 3 (mw 100) would result in which of the following?
(Note: the isoelectric point of each polypeptide occurs at pH 6)
Isoelectric point table
Polypeptide 1 pI=6
Polypeptide 2 pI=2
Polypeptide 3 pI=9

As written, polypeptide 1 has its pI at pH 6 but polypeptide 2 has its pI at pH 2 and 6 and polypeptide 3 has its pI at pH 9 and 6. A molecule cannot have multiple pIs.

 

[aldol16]

This is confusing I understand. But I wrote this question based on a KA question, which is posted by BeMd. My question and the original is different.

Okay, I'm still confused. No matter - I'll answer it both ways. If the question states that the pI of all the peptides is at 6 and the pH is 6, none of them will move because as I noted earlier, it's the electric field that causes motion. An electric field cannot act on something with no charge.

If the question states that the pIs are different, as in the original question posted (minus what's in the parentheses), then the negative one should migrate faster. That's because it's moving towards the positive electrode. The positively charged one would want to stay by the negative electrode. Weight doesn't matter there because weight only matters once the protein is moving. If it doesn't want to move, it could be the lightest thing in the world and still not move an inch. That's like dropping a ball into water. A ball with larger cross-sectional area will move through the water slower than one with a smaller cross-sectional area. But that's assuming that gravity acts on both balls. If gravity doesn't act on the smaller ball, it won't matter that it's smaller. In this analogy, gravity is the electric field. And of course, the uncharged one wouldn't move.

 

[weanprednisone]

So just ignore BeMD's question. That is not the question I asked. That is where we all got confused.
I've got it now, thanks @aldol16