Any articles or protocols related to the preparation of tissue lysates from freshly resected tumor samples for Western Blot would be appreciated
Dear Shivaani Srinivasan ,
I hope this protocol could be helpful.
Note: The entire preparation protocol should be conducted on ice.
1. Homogenize the fresh tissue samples with a motor-driven tissue homogenizer followed by filtration through a 70 μm cell strainer.
2. Centrifuge the filtrated homogenates at 200 × g for 10 min.
3. Remove the supernatant, and dissolve the pellets 5 ml hemolysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.2)
4. Then incubate it for 8 min, and wash at 200 × g for 10 min, again resuspended in 1 ml hemolysis buffer, and incubate it for 5 min.
5. After a final washing at 200 × g for 10 min, solubilize the pellets in PBS.
6. Dilute an aliquot till the final volume of 1 ml and transfer that to 8 cytospin cups (100 μl of suspension per cup).
7. Proteins from the samples are extracted by solubilizing the cell pellets obtained as described above in approximately ten sample volumes of lysis buffer (urea 8 M and Chaps 1% in PBS) for 30 min on ice.
8. Then centrifuge the samples at 10000 × g for 10 min at +4°C and use the supernatants for proteomics analyses.
I attached the related article for you.
Dear Sara Sheikhlary thank you for your answer. It was quite helpful
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