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Researchers discover novel method for detecting MIRCERA
Date:12/15/2009

nsity of the Dynepo and NESP bands gradually declined according to the decrease in their concentration, but a similar behavior was not observed for the MIRCERA bands. "We presumed from the experiments that the altered Western blotting performance of MIRCERA was due to its PEGylation (treatment with polyethylene glycol to obtain a long-acting effect of the protein)," noted Dr. Reichel.

PEGs of different average molecular masses (PEG 1500, 2000, 8000, 20000, 35000) were then separated on SDS-PAGE and visualized with a PEG-specific staining method. Due to a limited solubilizing power of SDS, PEGs regardless of molecular size were found to migrate as broad and smeared bands on SDS-PAGE. Given the results researchers hypothesized that the decreased sensitivity of MIRCERA on Western blots was due to an SDS-based solubility problem of MIRCERA's PEG-part.

To resolve the issue researches first used a detergent with higher solubilizing power for PEGs than SDS which did not improve the band shape of MIRCERA. Researchers then used SARCOSYL or (SAR)-PAGE with excellent results on Western blots by resolving MIRCERA in a sharp band and simultaneously keeping the resolution of rhEPOs and uhEPO on the performance level of SDS-PAGE. The authors found that "SARCOSYL was not binding to polyethylene glycol and was thus leaving PEGs in their native uncharged state." Redesigning SDS-PAGE by exchanging the SDS for SARCOSYL in the sample and running buffers solved the problem.

"In the past, there needed to be four methods performed to detect banned substances. Our method allows investigators to detect MIRCERA and other EPOs using just a single method," Dr. Reichel confirmed.


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Contact: Dawn Peters
physicalsciencenews@wiley.com
781-388-8408
Wiley-Blackwell
Source:Eurekalert

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