Evaluation of two high-abundance protein depletion kits and optimization of downstream isoelectric focusing

Qiu,Fanghua; Hou,Tieying; Huang,Dehong; Xue,Zhifeng; Liang,Dongyan; Li,Qiuming; Lin,Weimiao

doi:10.3892/mmr.2015.4417

November-2015 Volume 12 Issue 5

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November-2015 Volume 12 Issue 5

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Article

Evaluation of two high-abundance protein depletion kits and optimization of downstream isoelectric focusing

Authors:
- Fanghua Qiu
- Tieying Hou
- Dehong Huang
- Zhifeng Xue
- Dongyan Liang
- Qiuming Li
- Weimiao Lin
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Affiliations: Department of Clinical Laboratory, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong 510130, P.R. China, Department of Laboratory Medicine, Guangdong Academy of Medicine Science and Guangdong General Hospital, Guangzhou, Guangdong 510000, P.R. China, Department of Neurology, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong 510130, P.R. China
Pages: 7749-7755
|
Published online on: October 1, 2015

https://doi.org/10.3892/mmr.2015.4417
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Abstract

Disease biomarkers for diagnostic and prognostic purposes are most likely within an extremely low concentration range and are thus masked by the presence of high‑abundance proteins. Therefore, removing high‑abundance proteins is the main challenge for identifying disease biomarkers. In addition, the solution obtained from high‑abundance protein depletion kits contains a rich array of compounds, which interfere with isoelectric focusing (IEF). In the present study, the effect of two commercial kits was evaluated and the downstream IEF protocol was optimized. High‑resolution results could be obtained according to the following conditions: The ProteoPrep Blue Albumin and IgG Depletion kit depleted albumin and IgG; immobilized pH gradient strips (typically 18 cm) were rehydrated with sample buffer containing 250 µg serum proteins at 30 v for 6 h, 60 v for 6 h, 200 v for 2 h, 500 v for 2 h, 1,000 v for 2 h, 5,000 v for 2 h, 10,000 v for 2 h and then focusing at 10,000 v up to 110 k vhs. In addition, the protein spots identified by matrix‑assisted laser desorption ionization time‑of‑flight mass spectrometry demonstrated that all proteins had a low abundance. The present study not only provides a definite and effective method for removing high‑abundance proteins, but also provides a proper protocol (protocol C) for downstream IEF. The present study includes a comprehensive investigation of serum proteomics, which paves the way for serum protein research.

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Journals

International Journal of Molecular Medicine

International Journal of Oncology

Molecular Medicine Reports

Oncology Reports

Experimental and Therapeutic Medicine

Oncology Letters

Biomedical Reports

Molecular and Clinical Oncology

World Academy of Sciences Journal

International Journal of Functional Nutrition

International Journal of Epigenetics

Medicine International

Evaluation of two high-abundance protein depletion kits and optimization of downstream isoelectric focusing

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