Gel electrophoresis

[yellowjellybean]

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Hello,

1. I don't understand how the fragments are able to be separated if the bigger the fragment the more negative charge they have and so they have the same charge to mass ratio. Wouldn't they all move at the same rate then?

2. What is the purpose of the salt solution, is it to balance the charges like in a battery?

Thanks so much!

 

[Gauss44]

Hello,

1. I don't understand how the fragments are able to be separated if the bigger the fragment the more negative charge they have and so they have the same charge to mass ratio. Wouldn't they all move at the same rate then?

2. What is the purpose of the salt solution, is it to balance the charges like in a battery?

Thanks so much!

1. I gather that DNA fragments are separated by size. Amino acids are separated by charge. Is that right, anyone?

 

[dcamkl1]

Hello,

2. What is the purpose of the salt solution, is it to balance the charges like in a battery?

Thanks so much!

Is it really a "salt" solution? Usually electrophoretic gels are run using "running buffers" that contain ions that conduct the current.

For DNA gels, it's usually TAE or TBE, and for SDS-PAGE (protein) gels it's a buffer containing glycine, tris, and SDS.

1. I gather that DNA fragments are separated by size. Amino acids are separated by charge. Is that right, anyone?

Both are separated by size. With proteins, usually we denature them using SDS, which is an anionic detergent and gives the protein a uniform overall negative charge, so separating proteins on an SDS-PAGE gel is purely based on size. I can't explain this, but I do know that DNA has a uniform mass-charge ratio as well, so DNA is separated by size too.

The smaller ones will travel the fastest and will be further below. Bigger fragments will move slower and are towards the top.

An exception is a non-SDS gel (native gel) where the protein is not denatured using SDS, so the separation is based on size as well as charge.

Double check me though.

I think for the MCAT, the only time you would consider a native gel is when dealing with isolelectric points, where charge matters.

Otherwise, DNA and denatured protein (SDS) are separated by size.

 

[mehc012]

Is it really a "salt" solution? Usually electrophoretic gels are run using "running buffers" that contain ions that conduct the current.

For DNA gels, it's usually TAE or TBE, and for SDS-PAGE (protein) gels it's a buffer containing glycine, tris, and SDS.



Both are separated by size. With proteins, usually we denature them using SDS, which is an anionic detergent and gives the protein a uniform overall negative charge, so separating proteins on an SDS-PAGE gel is purely based on size. I can't explain this, but I do know that DNA has a uniform mass-charge ratio as well, so DNA is separated by size too.

The smaller ones will travel the fastest and will be further below. Bigger fragments will move slower and are towards the top.

An exception is a non-SDS gel (native gel) where the protein is not denatured using SDS, so the separation is based on size as well as charge.

Double check me though.

I think for the MCAT, the only time you would consider a native gel is when dealing with isolelectric points, where charge matters.

Otherwise, DNA and denatured protein (SDS) are separated by size.

The important thing to note about this is that protein conformation has an effect unless you denature them. 2 proteins of the same length could travel at different speeds if one has a more compact tertiary structure. This means you can't really standardize it the way you can DNA gels or when running denatured proteins...you can't look at a gel and say "this protein chain is longer than the other, because the band is higher", whereas you most certainly can do that with DNA (and why ladders can be used to determine DNA length).