I have to perform WB on FFPE tissue, so I used a protocol to extract the proteins but after the electrophoresis I got only one band.
This is the protocol I used
Extraction buffer (1 mL):
DTT 0,0031 g (200 mM)
SDS 0,02 g (2%)
Tris-HCl 1,8 μL (20 mM)
I reached the desired volume with distilled water
After deparaffination and rehydration I added the extraction buffer in the samples and incubated them at 99°C for 20 minuts and at 80°C for 2h. The surnatant obtained with centrifugation was stored in the fridge (3°C) for a couple of days. Then I just added Laemmli in 1:1 dilution and I incubated samples for 5 minutes at 95°C.
What could be the issue?
Thanks for your help
Dirk Schmidt
Thanks for your answer! Sorry for my ignorance, I'm a beginner and I actually didn't remember that I couldn't keep track of single proteins during electrophoresis. Anyway, is the protocol correct? This was my first attempt and I would like to fix any mistakes
Buon giorno Martina,
I cannot comment on the sample preparation as I never did that on FFPE-material. But I have my doubts about the gel and the Laemmli buffer. Please let us know how you prepared them (or is the gel precast?). What running buffers do you use? How is the Laemmli buffer composed?
What is the MW of the protein of interest? What % resolving gel was used? What pH do the various buffers have?
The blue tracking front stain should have been a narrow line. My guess would be the stacking gel has a pH that is too high (pH>6.8) or the resolving gel has pH
Duco Kramer Thanks for the answer! The gel could have some issues because it is expired, but the bands of standard protein solution are fine so I guess there's a mistake in sample preparation. Here there's another attempt with a further buffer (20 mM tris-HCl pH 8, 2% SDS).
I used a ready Laemmli buffer from Biorad, I only added to it 2-mercaptoethanol
I'm new in laboratory and I don't know what to fix, so you can say everthing that comes to your mind, even if it's something obvius, you'd really help me, although I think that the problem is in deparaffination and lysis of FFPE tissue
hi martina, Good that you shared the image of the gel. You say mol. weight marker (standard) is fine. I doubt that. The bands are not very sharp, while they should be. Sorry to disappoint you there! Here is an overview of all steps to follow when casting your own gels and the rest of the protocol. http://microbiology.ucdavis.edu/privalsky/sites/default/files/SDS-PAGE_0.pdf You work with precast gels (lucky you;) so you can omit the casting steps. Did you boil the protein sample after the addition of Laemmli buffer? The running buffer. Is that your 20 mM Tris HCL buffer? After you look into the protocol you may want to change it for a Tris-glycine buffer. It should be at the right pH after preparation. Did you order new gels? How long have they expired?
PS, I loved that Eurovision song festival song of Italy. Finally something that ROCKs! I admit, I love Italy as well ;)
Duco Kramer Thanks again! Yes, I know that the standard coloumn isn't exactly fine, but I mean, compared to what I obtained in the other coloumns and considering that the gel is expired, for me it's not that bad. Furthermore these are my first attempts, so I would be also glad to get some messy protein on my membrane :D
Yes, I boiled the protein sample + Laemmli. The buffer I described is for lysis, not for running. For running I use Tris-Glycine-SDS buffer
And yes, I ordered 10 new gels, they didn't arrive yet. But I want to try them only when I'm sure that I'm doing the other steps fine so I don't waste important material
And yeah, I'm happy they won such an important festival. It was a great opportunity to showcase our country's musicians and a great revival for the genre :3
Well, then we wait for results w/ fresh gels. Let us know if it works (or not...).
Duco
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