I have done SDS-PAGE for P.aeruginosa and its phage. I prepared 12% Gel and used 11-258 KD marker (Genedirex). There is a sharp band for marker but there aren't any bands for phage or bacteria.
I use sonicator to sonicate my samples and after that boil them with sample buffer for 10 min.
Does anyone have any idea?
Before cooking the samples in sample buffer, did you check how much proteins you got for each samples, and how much you loaded? Prolonged sonication can destroy pretty much all macro-molecules. Longer the more get destroyed. Try not to use sonication.
PS: Easiest way is to mix 1 vol your sample (P.aeruginosa and its phage without prior lysis) and 1 vol 2x SDS sample buffer or 3 vol sample + 1 vol 4x SDS sample buffer and load onto SDS PAGE for staining if you load enough total proteins. western blotting is more sensitive. SDS can make most of proteins released from bact. and denature them at the same time without heating. If you boil the sample it will make loading difficult since DNA will be release into the solution. Do a number of samples under different combinations of conditions you will know how to handle it and what is the best condition for your sample.
Most proteins do not need heating before SDS PAGE - western blotting (boiling for 10 min is harsh and some proteins, especially very large ones may not withstand this treatment). Alternative is 1-5 min at 70-90C.
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