Research Article
Comparability of a Three-Dimensional Structure in Biopharmaceuticals Using Spectroscopic Methods
Table 8
Troubleshooting for DTNB and DyLight 488 Maleimide methods.
| Problem | Cause | Solution |
| Native conditions | High variability on measurements. | Low protein quantity. | Increase protein concentration to 20–30 mg·mL−1. | Samples homogeneity. | Always vortex protein solutions. | High background. | Particles in buffer solution. | Filter buffer solutions through 0.2 μm hydrophilic membrane. | High sulfhydryl measurement. | Sample stress. | Avoid high temperatures and analyze samples within 12 hours once prepared. Dialyze samples at 4°C. | Sample excipients interference. | Dialyze samples against water. |
| Denaturing conditions | Low sulfhydryl measurement | pH lower than 6.5. | Verify buffer solution pH after GdnHCl addition. | High sulfhydryl measurement using DyLight 488 Maleimide. | pH higher than 7.5. | Verify buffer solution pH after GdnHCl addition. | Overincubation. | 2 hours of incubation must be enough for derivatization. | Incomplete dialysis. | Increase dialysis time and buffer exchange. |
| Denaturing-reducing conditions | Low sulfhydryl measurement. | Oxidized thiols. | Degasify all buffer solutions by sonication during 30 min and nitrogen bubbling during 2–15 min prior its use. | High background at 280 nm using DTNB. | TNB interference. | Estimate protein concentration prior derivatization. |
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