We need to accurately measure the protein/peptide concentration in a sample that is at least 80% DNA (primarily ssDNA less than 80 bases long with some dsDNA also). A majority of the methods I've looked at (Lowry, Bradford, NanoOrange, etc.) will not work with such a high DNA content. We also don't want to add any proteins (like DNAse) to get rid of the DNA because that will affect our measurement of protein contamination.
Any suggestions are welcome. Thanks!
How about Sodium Dodecyl Sulfate (SDS) Polyacrylamide Gel Electrophoresis (PAGE) analysis and staining with Coomassie brilliant blue R-250? It will only stain protein, not DNA, so the bands you see on the gel are all protein. You would see proteins of size 15kDa - 250 kDA using a 5-20% gradient gel or 12% gel might be sufficient.
Note: Constant Current Electrophoresis is much much quicker than constant voltage electrophoresis and the result looks good unless the SDS PAGE gel you are using is the size of a window pane because constant current heats up the gel with time.
You could load Bovine Serum Albumin (BSA) and Lysozyme of known concentration and quantity to separate lanes of your gel, as well as your potentially contaminated samples. Analyze your gel on a gel imager and save the "*.gel" file and analyze it in ImageJ. Draw a box and measure the intensity with CTRL + M in ImageJ. You know the quantity of BSA and lysozyme you loaded to the gel so calculate the intensity and assign that to the quantity of the proteins. You could make a standard curve of ImageJ intensity of BSA and lysozyme vs quantity if you load different quantities of each protein to the gel.
Raw intensity data is encoded in "*.gel" files. "*.png" and "*.jgp" files just contain pixel information.
A brief look at a Coomossie stained SDS PAGE gel will tell you if your samples are obviously contaminated with protein. ImageJ intensity analysis of the gel with BSA and lysozyme standard curves of imageJ pixel intensity vs protein quantity should tell you the range of protein quantities for your other proteins in your DNA sample contaminated with proteins. Since your detecting quantiity if you don't know the concentration of your samples, you should know the volume you loaded into the gel. If drawing a box is too painful ImageJ has other fancy tools.
Detection limit for Coomossie Brilliant Blue R-250 seems to be about 1 migrogram of protein. If that's not sensitive enough for you then G-250 seems to be more sensitive. Silver staining is more sensitive than the others, but the disadvantage is that silver stains DNA and also proteins.
You could also consider mass spectrometry. Intact mass spectrometry can detect the mass of all proteins in a sample without fragmentation if the proteins ionize well. Most mass spec systems are setup to detect only positive charges and since DNA is heavily negatively charged, it will be invisible to the detection. Mass spec is very sensitive to low quantities of protein, but the more protein you have the better the detection as there is more to detect.
Bottom up Liquid chromatography with MS/MS and trypsinization can detect the mass of all peptide fragments after trypsinization of peptides and proteins, but deconvolution of what those peptides correspond to is kind of catch22 because you have to know the sequence of the proteins in your sample. Nevertheless, it will detect all peptide fragments.
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