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Molecule 101: Urea (1)

Urea, this small molecule has been a lifesaver in my lab work, especially when dealing with stubborn western blots for transmembrane proteins. Urea, composed of two amide groups (Figure 1), is one of the most widely used protein denaturants in protein biochemistry. 


Fig 1. Chemical structure of Urea

(Fun fact: if you’re looking for smoother hands, check out hand creams containing urea—it’s also a moisturizing agent!)

One of urea’s favorite applications is in proteomics, where it is used to unfold and solubilize proteomes, making them susceptible to trypsin digestion. Its history in protein science goes back to Anfinsen’s famous experiment, which demonstrated that the code for protein folding reside in the amino acid sequence. Anfinsen unfolded ribonuclease using urea, and upon removing it, some proteins refolded, proving the “code” for folding is intrinsic to the sequence.

Like many scientists, I’ve been using urea in my experiments for years without giving much thought to how it actually works. So, let’s dig deeper into the question:

How Does Urea Work?

This question has intrigued protein chemists for decades. After diving into the literature (PNAS, PubMed ID: 17503819, PubMed ID: 20121105, ACS Publications), it seems like urea denatures proteins through a combination of direct interactions and indirect interactions (although direct interactions are more acknowledged): For direct interactions, Urea forms hydrogen bonds with the peptide backbone and side chains while also engaging in electrostatic and van der Waals interactions. For indirect effects, Urea disrupts water networks, reducing the hydrophobic effect and stabilizing solvent-exposed conformations. (My visual cue for urea’s action is that it seems to ‘tear apart’ the intricate network of intramolecular interactions holding the protein’s folded structure together.)

These mechanisms work together to destabilize native protein structures and shift the equilibrium toward unfolded states. Of course, urea’s carbonyl group and amide groups play a critical role in these direct interactions.

Practical Use: Urea in Transmembrane Western Blots

Transmembrane proteins, with their arrays of hydrophobic helices, are notoriously difficult to analyze using SDS-PAGE or western blotting. Standard sample preparation methods—boiling proteins in SDS at high temperatures—often fail for transmembrane proteins. Instead of fully denaturing, the proteins aggregate or remain in a limbo state ( I guess somewhere between aggregation and unfolded state?), causing smears and poor resolution in blots.

Here’s an example from my own experiments: in the left panel of my blot (Figure 2), you can see a giant smear where my YFP-tagged transmembrane protein should have been resolved. After incorporating urea into my protocol, the right panel shows a clear band corresponding to the expected protein size.

   Fig 2. My transmembrane blot 


The recipe of protocol is simple: Prepare your protein sample with a final concentration of 4 M urea and 5% beta-mercaptoethanol. Then, incubate at varying temperatures (room temperature, 37°C, and 70°C are good starting points for optimization).



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