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Failed BCA protein assay, white crystalline solid after speedvac - (Mar/01/2012 )

Hi there

I was wondering if anyone might have a clue to some weird happenings in my recent western blot.

I acquired some adipose tissue samples recently and homogenised the samples with SHE buffer added with protease + phosphatase inhibitor. Forseeing that the amount of protein might be limited, we only used 200ul of SHE buffer for homogenisation. I spun it at 12,000 RPM for 15 mins and aliquoted out the supernatant.

I tried to do a standard BCA to measure the proteins in the supernatant but the colour in the assay wells turned copper sulphate blue with the samples instead of purple or green. My BSA standards looked fine.

I then tried a Bradford protein assay (never done it before) and got some readings. I observed that the protein concentrations were very low, around 80-200 ug/ml.

I then used the speedvac (Hetovac) to concentrate the samples to about 1/4 of its volume. A white crystalline precipitate appeared in my concentrated samples. I thought it might be lyophilised protein so I went ahead with sample loading preparation. I added my dye and boiled the solution for 5 minutes. The white precipitate did not dissolve on boiling and on loading. It did not take the blue dye either.

On running the gel we saw this happen. (See attached picture) Leftmost is my marker, which was affected by the next two rows which was the tissue samples. The six rows to the right are my cell lysates which migrated fine.

Any idea as to what these white solids might be? Nothing is known about the sample handling before it came to my lab but it was meant to be WB/PCR friendly samples.

We've consulted with people around the departments and all of them haven't seen anything like this in all their (many)years of experience.
Attached Image

-Cherlyn Ding-

since you are using adipose tissue, i would guess that it is caused by lipids.

the white precipitate would not be lyophilized protein, you didn't go to dryness. it could be lipid or carbohydrate (or lipopolysaccharide?). either will disrupt electrophoresis of proteins.