I have to perform Western Blot on FFPE tissuse
First I did deparaffination, then I added the lysis buffer (20 mM Tris-HCl pH 8, 2% SDS) and I incubated for 20 minutes at 99°C and for 2h at 80°C.
I ran the gel for 30 minutes at 200 V
I transferred overnight (12h) at 10 mA
The pictures here are what I got after running and after the blotting, with apparently no protein at all (I stained with ponceau S)
The gel is expired so I know that is normal that it isn't perfect, but with the protein standard solution the bands are neat. So there are clearly some issue in the steps before. What could it be? Is it a buffer problem or something about deparaffination? Can you tell me?
I'm a beginner in laboratory, so you can say everthing that comes to your mind, even if it's something obvius
Thanks for your help
Can you provide some more details on the tissue that you used for this analysis:
- What kind of tissue?
- How much tissue was used?
- Was the tissue homogenized before lysis?
Hello Martina Boncaldo
I agree with John Hardy Lockhart . The information provided is incomplete. It seems to me that there is a problem with sample extraction. It is not easy to work with FFPE tissue. You need to optimize the protocol.
Regards
To add to the answers already provided, the key issue here is likely cross-linking. Your FFPE tissue is not just embedded in wax (which deparaffinization can remove) but is also really quite heavily formalin-fixed.
Formalin crosslinks proteins to each other, and does so essentially non-specifically, and that's not going to work with SDS PAGE. Boiling alone is not generally sufficient to break all those crosslinks (which is why HIER protocols usually also include citrate).
This paper Article Efficient extraction of proteins from formalin-fixed paraffi...
seems to suggest that at high concentration tris itself can act as a cross-link scavenger, so you might want to explore that: high tris (300mM+) concentrations plus the prolonged incubations at high temperatures that you're already doing.
John Hardy Lockhart Yes, here's the information you need:
- It's dog breast cancer tissue
- About 5 sections 5 μm thick
- Tissue was already sliced when I performed deparaffinization, so I guess that the pellet I got was pretty omogenized: the protocol that I followed didn't say to do something for this step. Should I have to do something about it anyway? What do you suggest?
Malcolm Nobre My previous answer provides the information requested. Do you have any suggestion for the optimization of my extraction protocol?
John Hildyard Thanks for the answer! I will try to do some attempts adding more Tris in buffer preparation.
I wanted to try this buffer: 200 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5% SDS and 100 mM sodium citrate. Do you think that using this will make the bands after SDS-PAGE neater?
- Badges
- Science method