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.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression and clinical significance of KiSS- 1 and E- cadherin in gastric cardia carcinoma and the correlation between the two proteins.</p><p><b>METHODS</b>The expression of KiSS- 1 and E- cadherin in 80 patients with gastric cardia carcinoma and 20 patients with normal gastric cardia epithelium was detected by immunohistochemical technique.</p><p><b>RESULTS</b>The expression of KiSS- 1 was negatively correlated with lymphatic metastasis and clinical stage (P < 0.05), but not correlated with the cancer differentiation (P < 0.05). The expression of E- cadherin was negatively correlated with lymphatic metastasis, clinical stage, and cancer differentiation (P < 0.05). Spearman test showed a positive correlation between KiSS- 1 and E- cadherin expression (r(s)=0.722, P < 0.05).</p><p><b>CONCLUSION</b>KiSS- 1 and E- cadherin may play important roles in inhibiting the invasion and metastasis of gastric cardia carcinoma.</p>