What method we should use to separated the two proteins from the mixture in which one is monomeric with
MW of 10 kDa and other is of two Homodimer’s with MW 10 kDa and 15 and that are

(a) Nonreducing SDS-PAGE

(b) Reducing SDS-PAGE

(c) Native PAGE

(d) All of the above

Correct answer: (c) Native PAGE

Explanation:

The key difference between Native PAGE and SDS-PAGE (reducing or non-reducing) is that Native PAGE maintains the proteins in their native, folded, and oligomeric states.

In the given mixture:

1. Monomeric protein (MW = 10 kDa)
2. Homodimeric protein (MW = 10 kDa + 15 kDa)

Why Native PAGE is the best choice?

• No denaturation: Unlike SDS-PAGE, Native PAGE keeps proteins in their natural state, preserving quaternary structure (i.e., the homodimer remains intact).
• Separation by size and charge:
  • The monomeric protein (10 kDa) will migrate based on its charge and shape.
  • The homodimer (10 kDa + 15 kDa = 25 kDa) will migrate differently due to its larger size and different charge distribution.
• This allows separation based on their natural form rather than just molecular weight.

Why Not the Other Options?

1. (a) Non-reducing SDS-PAGE ❌

   • SDS denatures proteins, but without a reducing agent, disulfide-linked dimers remain intact.
   • The homodimer (25 kDa) and a single 25 kDa protein would appear the same, making differentiation difficult.

2. (b) Reducing SDS-PAGE ❌

   • A reducing agent (β-mercaptoethanol or DTT) would break disulfide bonds, splitting the dimer (10 kDa + 15 kDa) into two separate bands (10 kDa and 15 kDa).
   • This would not allow us to observe the homodimeric form, which is what we want.

3. (d) All of the above ❌

   • SDS-PAGE (both reducing and non-reducing) denatures proteins, which is not ideal for separating native monomeric vs. dimeric forms.
   • Only Native PAGE can separate them in their biologically relevant forms.

Conclusion:

Since Native PAGE preserves protein structure, it is the best method for distinguishing monomeric (10 kDa) and dimeric (25 kDa) proteins in their native states.

✅ Correct answer: (c) Native PAGE