Hey all, I'm very interested in understanding more about the biochemistry behind all of the wash buffers that go into the streptavidin affinity purification protocol seen here (https://static1.squarespace.com/static/5617d7d8e4b09f2fdf34baa6/t/5fe2631ba775596e136098b6/1608672031257/Cho+NP+2020.pdf pg 3990, step #38).
I understand this is to help reduce non-specific interactors from being pulled down with the biotinylated proteins; however, I'm more curious to understand the necessity of the 2M Urea vs sodium carbonate vs KCl, etc....looks like previous iterations of this protocol have not used sodium carbonate and KCl buffers but I can't find substantiation for why they were added.
Thanks in advance!
The sodium carbonate is acting as a buffer to maintain the pH as some wash buffer formulations use phosphate instead. The salt is used to increase the ionic strength of the buffer to reduce intermolecular interactions in the assay which results in a reduced number of false positives.
hello dear Sarah
The purpose of the wash buffers is to minimize non-specific interactions during the purification process.
Here is a detailed and scientific explanation of the different components in the wash buffers and their roles:
1. **2M Urea**: Urea is a chaotropic agent, meaning that it disrupts water's hydrogen bonding network and can cause proteins to lose their native structure. In this protocol, 2M Urea is used as a mild denaturant to help reduce non-specific protein-protein interactions. This concentration of urea is not strong enough to completely denature proteins but can help minimize weak and non-specific interactions, which is essential for the purification of specific protein complexes.
2. **Sodium carbonate (Na2CO3)**: Sodium carbonate is a mild alkaline buffer that can help maintain pH during the purification process. This buffer can also promote protein solubility by neutralizing acidic residues on the protein surface. Additionally, its ionic nature may help with the disruption of non-specific electrostatic interactions, further improving the purification process.
3. **KCl**: Potassium chloride (KCl) is a salt that can be used to modulate the ionic strength of the buffer. High ionic strength can help reduce non-specific interactions by weakening electrostatic interactions between proteins. The presence of KCl in the wash buffer can help improve the specificity of the streptavidin-biotin interaction by reducing the likelihood of non-specifically bound proteins remaining associated with the streptavidin resin.
It is possible that previous iterations of the protocol did not include sodium carbonate and KCl buffers because they were not deemed necessary for specific applications or because the researchers were using different buffer systems. However, the inclusion of these components in the wash buffers can improve the stringency of the purification process, increase the specificity of protein complex capture, and decrease background noise in downstream analyses.
Just note that sodium carbonate Na2CO3 is NOT a buffer. To work as buffer, you'd need some acid to complement it (e.g. H2CO3 or NaHCO3). This is "just" alkaline solution used to dissociate some interactions.
For example, I have used sodium carbonate when I have fractionated proteins associated with cell membrane few years back.
Hi,
The KCl helps remove some non-specific target by releasing them from potential membrane remanent (not sure).
Urea and Na2CO3 slightly denature proteins on beads that only biotinylated protein remain on the beads but not other proteins interact with biotinylated proteins.
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