Background and Objectives Two-dimensional SDS PAGE (sodium dodecyl sulfate polyacrylamidegel electrophoresis) coupled with mass spectrometry is still a mainstream approach to analysing multiple protein expression levels. The requirement for some sophisticated devices and the lack of quantitative measurements for low-abundant proteins (e.g. cytokines) greatly limit its broad application. Cytokines present in the pg/ml levels in non-stimulated biological samples are traditionally detected by ELISA. We used a cytokine antibody array, a highly sensitive protein chip, for simultaneous detection of multiple cytokine expression levels in rat sarcoma lysates and serum samples . Material and methods We present here an optimized protocol for preparation and handling of tumour tissue lysates in protein chip detection. The sarcoma samples were processed at low temperatures to prevent cytokine degradation. Tumour cryosections (8?10 mm) were used for extraction of cytokines. The addition of NaN3 destroyed a high endogenous peroxidase activity, which may interfere with protein chip assay and decrease the signal/noise ratio. The data for the protein matrix effect from sandwich ELISA can also affect the protein chip detection. The optimal dilution of samples must be found to prevent pitfalls due to the non-optimal signal-to-noise ratio. This also enables recovery of low amounts of cytokines from difficult samples. Results We report optimized procedures for extraction, sample handling, inhibition of endogenous peroxidase activity and prevention of the protein matrix effect in serum and tumour lysates by detection of cytokine expression using the cytokine antibody array protein chip.

Ústav živočišné fyziologie a genetiky AVČR Liběchov

Protein chips in cytokine expression study on tumor model: optimalization of sample preparation

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