we tried to isolate and western blot proteins from Formalin fixed paraffin embeded tissues. We did this protocol:
1. we got 5 x 10 micro meter paraffin sections.
2. xylene, 96-90-80-70 alchols, distile water and then put in
3. % 2 SDS in RIPA buffer in 100 oC 20 mins then 60oC 2 hours.
4. 15.000 g, 4oC, 20 mins centrifuge.
5. Protein measurement by Qubit 2.0, then western blot protocol.
At the end, we didnt get any bands.
What can you offer us?
I can see some faint bands and lanes clearly. I guess your protein yield is too low to detect. Are you detecting specific proteins or total proteins? Did you check the transfer with Ponceau S? Need more details to help you better. Goodluck
the protein ladder is in the right side. We searched beta-actin in human thyroid papillary cancer tissue. We checked with Ponceau S and unfortunately we couldnt see any bands :(
I assume you need to concentrate your sample and load more protein, which might solve your problems. To have a kind of positive control for you western blot, you better load normal cell or tissue lysate (human or mouse, whichever matches with your sections) so that you will know if there are additional problems with transfer, antibody and exposure problems. Goodluck
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