Hi Colleagues,
I just did a western today to look at Phospho Tau and Total Tau protein levels in hippocampus of 3TgAD mice and met some complications
[Phospho Tau Pierce MN #1020; Total Tau BD556319]
I didn get any staining for Total Tau!?
I am trying to figure out why.
The Tau protein can be present in soluble and insoluble forms. The soluble form is found in the supernatant of the homogenate, and the insoluble Tau in the pellet, dissolved in formic acid.
[The tissue was homogenized in RIPPA with protease inhibitors (including phosphatases)]
I run the samples to test for soluble Tau.
First, I stained for Phospho Tau, got a very strong signal in 3 samples.
Then probed for Actin, which looks fine, overloaded, but fine
I, then, stripped the blot with b-me containing buffer (to probe for T Tau) and stained with Ponceau S to make sure there is still some protein left.
The Ponceau looks fine
I proceeded to probe for Total Tau, but when I develop the blot I have no signal
There is a dark spot that comes up from long incubation with ECL. I am not 100% sure what it is, but I think it is just from over exposure of the blot
I was pretty certain to see something for Total Tau.
What would cause this?
I am thinking it is a problem with the primary antibody or maybe it is tihe unfavorable blocking/primary incubation buffers?
Does anybody ever had this problem and how did you fix it?
I loaded 40ug of protein. We should be seeing something, I hardly think that there is not enough protein left to be tested
Any suggestions would be highly appreciated
Thank you all
You guys are lifesavers and absolutely the best!!
P.S.I included some images below
Total tau in the soluble fraction of 3xTg-AD mice should definitly work. Sometimes, the simplest answer is the better. Did you test you antibody first on 1-2 brain samples without stripping, to make sure it works properly and that you have optimized the blotting conditions (buffer, antibodies concentration, etc)? Some antibodies simply won't work after stripping. Usually, these kind of problems are the results of errors. Before trashing your membrane, double ckeck that you blocked the membrane after stripping, used the correct secondary antibody for the detection, etc. The use of different species for antibodies against phospho-tau vs. total tau makes it possible to probe twice without stripping.
Good luck!
- Badges
- Science method