SDS PAGE

[Astra]

My understanding is the following:

SDS page is a form of electrophoresis where the proteins' hydrogen bonds are disrupted leaving behind the primary structure of a protein to be assessed by its molecular weight.

Non reducing SDS page does not disrupt the hydrogen bonds and this allows a protein to have its tertiary/ quaternary structure.

Why would non reducing SDS page cause a homodimer to breakdown into 2 monomers but not affect a tetramer linked via sulfide bonds?

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[aldol16]

SDS is sodium dodecyl sulfate and a detergent. What that does is 1) disrupt the H-bonding such that the protein denatures and 2) coats the protein with a lot of negative charges so that they all migrate towards the anode on the gel.

Non-reducing gels have nothing to do with H-bonding. Non-reducing gels are normally run in the presence of SDS. Perhaps you're thinking of native gels. You should always keep chemistry in the back of your mind and try not to compartmentalize. The key word is non-reducing. What does that mean? The answer to this question will also answer your third question.

 

[theonlytycrane]

a simple example: we have a protein of interest and want to analyze its composition. Say we know it weighs 100 kDa.

Run SDS page (non-reducing gel). This denatures the protein to disrupt tertiary structures, but leaves disulfide bonds intact. Say from the gel we interpret that there are 2 subunits- one of 60 kDa and one of 40 kDa.

Run SDS page one more time (reducing-gel). This time the protein denatures and disulfide bonds are broken. Say from the gel we still see the 60 kDa mark but now see a 20 kDa mark instead of 40 kDa.

We can interpret that the protein is composed of 3 subunits. Two are 20 kDa are connected via disulfide bond(s). The other is 60 kDa and interacts with the other units via non-covalent interactions.

For your question: if a non-reducing page causes a homodimer to breakdown, it must be because the homodimer is not connected via disulfide bonds.

 

[Astra]

a simple example: we have a protein of interest and want to analyze its composition. Say we know it weighs 100 kDa.

Run SDS page (non-reducing gel). This denatures the protein to disrupt tertiary structures, but leaves disulfide bonds intact. Say from the gel we interpret that there are 2 subunits- one of 60 kDa and one of 40 kDa.

Run SDS page one more time (reducing-gel). This time the protein denatures and disulfide bonds are broken. Say from the gel we still see the 60 kDa mark but now see a 20 kDa mark instead of 40 kDa.

We can interpret that the protein is composed of 3 subunits. Two are 20 kDa are connected via disulfide bond(s). The other is 60 kDa and interacts with the other units via non-covalent interactions.

For your question: if a non-reducing page causes a homodimer to breakdown, it must be because the homodimer is not connected via disulfide bonds.

SDS is sodium dodecyl sulfate and a detergent. What that does is 1) disrupt the H-bonding such that the protein denatures and 2) coats the protein with a lot of negative charges so that they all migrate towards the anode on the gel.

made perfect sense Thanks a lot!

So in the case of reducing gels, they disrupt only sulfide bonds? that means they leave in tact

1. hydrogen bonds between polar R- groups
2. ionic bonds between charged R-groups
3. hydrophobic interactions between nonpolar R-groups

 

[aldol16]

So in the case of reducing gels, they disrupt only sulfide bonds? that means they leave in tact

1. hydrogen bonds between polar R- groups
2. ionic bonds between charged R-groups
3. hydrophobic interactions between nonpolar R-groups

No, because reducing gels are usually run in the presence of SDS, which is a detergent that disrupts H-bonding and ionic contacts, and under denaturing conditions. To my knowledge, nobody runs a reducing native gel.