I have tried to detect the difference in NKCC and CFTR between two regions of an eye with Western Blot. There was no problem for the detection of NKCC. For CFTR, very light or even empty bands were found. I wonder whether the sample amount was too small, so I pooled the sample from two eyes together. However, the results were almost the same.
CFTR expression in some organs is low, if in eyes also is low expression maybe you can increase the protein concentration by freeze dry, or aceton percipitation
OK, but what was the quantity loaded on the gel? How many microliters did you use?
OK, so you are loading 9-14 ug on your gel, I do not know the CFTR expression level in RPE, but the quantity you load seems low.
Like Khadijeh said, you can try to concentrate your proteins. A simple way is to add 1.5 vol of -20°C ethanol to your proteins, incubate at 4°C for 30min, centrifuge at 16000g for 15min at 4°C, discard the supernatent, and resuspend the pellet in your protein extraction buffer.
You may also try to immunoprecipitate CFTR on larger volumes.
Sylvain and Khadijeh, thank you for your advices.
I'll try to concentrate the samples according to your suggestions.
Concentration may be one of the questions, another question is the antibodies.
We don't have anti-chick antibody, what we used is anti-mouse (1:1000).
Would it be another cause of the failure?
Sure! Your antibody may recognize mouse CFTR but not chick CFTR. You better use an antibody known to recognize chick CFTR.
Unfortunately, there is no antibody available for chick CFTR.
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