Is it okay to run reducing SDS-PAGE and non-reducing SDS-PAGE on the same gel?
It is not recommended to run reducing SDS-PAGE and non-reducing SDS-PAGE simultaneously on the same gel for the following main reasons:
1. Diffusion of reducing agents affects non-reducing lanes
1. Reducing SDS-PAGE typically involves adding DTT (Dithiothreitol) or β-ME (β-mercaptoethanol) to break disulfide bonds and fully denature proteins.
2. However, reducing agents may diffuse into non-reducing lanes during electrophoresis, partially affecting non-reduced proteins, leading to abnormal bands.
2. Different protein migration rates
1. Under reducing conditions, proteins are fully denatured and migrate based on molecular weight.
2. Under non-reducing conditions, proteins may retain higher-order structures formed by disulfide bonds, resulting in different migration patterns.
3. Such mixed migration can make it difficult to accurately compare protein bands.
3. Inconsistent electrophoresis environment within the gel
1. Reducing agents may affect the buffer pH, causing uneven electrophoresis conditions across different lanes on the same gel.
2. Reducing agents may affect protein staining, diminishing the comparability of bands.
4. Best Practices
1. Run two separate gels for reducing and non-reducing SDS-PAGE to obtain accurate protein band information.
2. If running on the same gel is necessary, it is recommended to:
- Place reducing samples far from non-reducing samples (though diffusion may still affect them).
- Reduce the amount of reducing agent used, although this may impact complete protein denaturation.
- Complete electrophoresis quickly to minimize the diffusion time of reducing agents.
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