Comparison of different protocols for protein extraction from formalin-fixed paraffin embedded esophageal squamous cell carcinoma tissues
10.16098/j.issn.0529-1356.2021.01.023
- Author:
Ye-Lin JIAO
1
;
Hao-Jie RUAN
1
;
She-Gan GAO
1
;
Yi-Jun QI
1
;
Ye-Lin JIAO
2
;
Yun-Gang ZHAO
3
;
Qi-Wei LIU
4
Author Information
1. He'nan Key Laboratory of Cancer Epigenetics, Cancer Hospital, The First Affiliated Hospital, College of Clinical Medicine, Medical College of He'nan University of Science and Technology
2. Department of Pathology, The First People's Hospital Luo Yang
3. Department of Medical Genetics, Hospital for Maternity and Children's Healthcare of Changzhi City
4. Department of Anatomy, Xuchang University
- Publication Type:Journal Article
- Keywords:
Formaldehyde-fixed;
Paraffin-embeded tissue;
Protein extraction;
Protein purification;
Western blotting
- From:
Acta Anatomica Sinica
2021;52(1):141-145
- CountryChina
- Language:Chinese
-
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 °C or 105 °C. 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 °C 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 °C 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.
