I isolated endosome from SH-SY5Y after treated. Western blotting after BCA. The targeted protein changed a lot. And the problem is the internal control (EEA1) is significant different between groups. The group with higher my targeted protein expression will have a lower EEA1 expression and vice versa. I've repeated BCA for about 5 time and confirmed the protein concentration I loaded are the same.
How can I deal with the situation?
Sebastian Schmitt
Yes, I'm sure I've got the same amount of total protein for each sample. I confirm the point for several times. I'm now thinking about if the changes of my targeted protein is so big that might affect the loading controls within the same total protein.
I recommend that you try out Total Protein Normalization reagent. Ponceau S staining is cool but during the rinsing step, a part of your blot might be washed off quicker than another part and this might raise further questions.
Hi,
I had once seen advertisement from biorad for TGX stain free protein labeling and UV visualization in the gel or on the membrane after transfer.
It seems you obtain different amounts of early endosomes (EEA1+ vesicles) depending on your conditons and you look for your protein of interest in this preparation.
In this case, the question would also be at which level do you normalize your signal
- Equal starting material in terms of mg whole cell extract for endosomes isolation
- Equal amount of isolated vesicles regardless of whether a condition contains more or less of them.
And also the proportions of endosomes types in the preparation.
I don't know with which technique you isolate the endosomes, for example if it is by ultracentrifugation on a gradient is EEA1 that is specific of early endosomes a good loading control for a whole endosomes preparation (early to late, maybe even mutivesicular bodies)?
Best,
Daniel
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