So these are my saline-treated cerebellum samples. Lanes 1-9 are loaded with equal amount of protein. This membrane was stained for b-actin only, and as you see some bands are missing (my protein of interest, which is 96kDa is absolutely fine). What happened to some of my samples? Transfer looks fine
Could it be that lower MW protein just went missing during homogenisation/heat treatment?
For reference, all samples were processed on the same day using the same buffers. Also, my treated samples are absolutely fine.
10ug protein per well
5% milk
beta actin 1:5000
seconday Ab 1:2000
Thanks
You see a good relationship between the stained samples et the immunoblots. The problem could have happened when loading the gel. Precipitation of proteins in your tube, leak in the well? You don't have 10 ug of protein in all the wells.
Thank you for your opinion Genevieve,
What would you suggest me to do? Is it worth trying to do a Bradford assay on my electrophoresis samples to re-estimate the protein concentrations, so that I can load equal amounts in the wells next time?
thanks
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