Comparison of different protocols for protein extraction from formalin-fixed paraffin embedded esophageal squamous cell carcinoma tissues

doi:10.16098/j.issn.0529-1356.2021.01.023

Abstract

Abstract:

Objective To explore protein extraction efficiency from formaldehyde-fixed paraffin embedded (FFPE) esophageal squamous cell carcinoma (ESCC) tissue samples with different protocols. Methods Six different lysis buffers with 100 ℃ or 105 ℃ treatments were used for protein extraction, followed by evaluation of protein quantity and quality with Bradford, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, Western blotting and immunohistochemistry (IHC), using 8 FFPE samples of ESCC. Results The optimal method for protein extraction from FFPE ESCC tissue was Laemmli lysis buffer (Buffer 4) treated with 100 ℃ incubation, evidenced by highest amount of protein recovery. Western blotting and IHC method measured consistent 14-3-3σ expression in FFPE ESCC tissue samples. Protein precipitated by two volumes of acetonitrite acetonitrile(ACN) (0.1% trifluoroacetic acid) relative to protein amount reduced background staining on SDS-PAGE gels by commassie staining. Conclusion Laemmli lysis buffer combined with 100 ℃ incubation has the highest protein extraction efficiency from FFPE ESCC tissue samples for Western blotting measurement of protein biomarkers, and ACN protein precipitation can further eliminate residual cross-linked protein by FFPE.

Key words: Formaldehyde-fixed, Paraffin-embeded tissue, Protein extraction, Protein purification, Western blotting

CLC Number:

R-735.1

JIAO Ye-lin ZHAO Yun-gang LIU Qi-wei RUAN Hao-jie GAO She-gan QI Yi-jun . Comparison of different protocols for protein extraction from formalin-fixed paraffin embedded esophageal squamous cell carcinoma tissues[J]. Acta Anatomica Sinica, 2021, 52(1): 141-145.

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