Comparison of different protocols for protein extraction from formalin-fixed paraffin embedded esophageal squamous cell carcinoma tissues / 解剖学报
Acta Anatomica Sinica
; (6): 141-145, 2021.
Article
в Zh
| WPRIM
| ID: wpr-1015512
Ответственная библиотека:
WPRO
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.
Полный текст:
1
База данных:
WPRIM
Язык:
Zh
Журнал:
Acta Anatomica Sinica
Год:
2021
Тип:
Article