Silvia Costas Abalde, University of Santiago de Compostela
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Applications: Western Blot
Species: Mouse
Sample: Skeletal muscle (gastrocnemius, diaphragm, soleus), heart, Pancreas (healthy and tumor)
Experimental Conditions: WB: Lysate in ice-cold RIPA (50 mM Tris-HCl pH 7.2, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, protease/phosphatase inhibitors); 20 µg protein; 15% acrylamide gel; transfer nitrocellulose tank blot 1 h 250 V 0.33 A, Ponceau S check; blocking 5% BSA 1 h RT; primary 1:1000 O/N; wash 3×5 min TBST; secondary Jackson 111-035-003 1:30,000 1 h; detection Pierce ECL, ChemiDoc MP.
Dilution: WB: 1:1000
Results Summary: Detected two clear bands in gastrocnemius, soleus, and heart: ~15 kDa (likely ghrelin) and slightly above 15 kDa (likely preproghrelin). Pancreas and diaphragm showed more non-specific bands but also two well-defined bands at ghrelin/preproghrelin positions. Bands consistent with expected MW for target proteins.
Comments: Good specificity in muscle and heart tissues; more background in pancreas and diaphragm.
