I am trying to look at the expression of c-fos nuclear protein in mice brain using WB technique.
Can anyone suggest a protocol to extract the proteins? Better harsh homogenate before sonication? Should I use the Tris-HCl Buffer or RIPA is better? What ratio between lysis buffer and tissue weight?
Many thanks,
Ivan
I recommend you not to use ultra sonication, since it may denature the proteins within your samples. Dounce homogenizer is the best apparatus for homogenizing brain tissues. Tris Buffer or RIPA, I recommend you adding a protease inhibitor cocktail to them. A 1:10 ratio (sample: lysis buffer) is good according to my experiences.
Hello, Ivan
About choosing the Lysis buffer, I believe it depends on which protein(s) you are interested. Tris-HCl buffer is commonly used for cytoplasmic protein extraction, and RIPA buffer is usually used for membrane-bound or nuclear proteins. As you said you are interested in nuclear protein, I would recommend RIPA buffer for protein extraction.
You can check more details for chosing a proper lysis buffer in the Abcam website (https://www.abcam.com/protocols/sample-preparation-for-western-blot#lysis-buffer).
About the tissue:buffer ratio, 5 mg of tissue can be homogenized with 300 uL of lysis buffer, but you can add more if you feel the protein is still too dense.
For the homogenate preparation, I usually use a dounce homogenizer and do it manually.
Good luck!
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