For particularly difficult samples, be sure to start with samples that are as finely pulverized as possible. Especially for tough samples like tissues, either find and use a bead beater under liquid nitrogen, or use a Covaris cryoPREP to pulverize it. Alternatively for larger scales and absolute maximum pulverization, dice your sample (preferably frozen), lyophilize it and use a laboratory-scale jet mill. Note the losses here will be higher.
If you are sample limited and/or want to improve your recovery, use a Covaris AFA unit which is well suited to small sample amounts, or a cryogenic bead beater with frozen 5% SDS. For AFA, place the sample in 5% SDS and immediately AFA treat until the sample is fully disaggregated. Samples are kept isothermal typically at 4 C. For cryogenic bead beating, freeze 5% along with the sample, make sure the bead(s) are able to move, and pulverize until a free-flowing powder results. In both cases, 5% SDS will coat the surfaces of tubes, tips and beads, limiting loss. Surfaces can be washed if desired with additional buffer.
If a Covaris AFA unit or cryogenic bead beater is not available, probe sonication is an option. Be careful, however, to not explode your sample out the top of the tube. Alternatively or additionally, incubate samples with agitation at elevated temperatures (e.g. 55 C for 2 hrs on a heating block or overnight at 37 C on an end-over-end rotator). Note that elevated temperatures can cause sample degradation, even with enzymes like proteases and phosphatases inhibited by 5% SDS.
For tissues and other samples which may be crosslinked through disulfide bonds, we recommend including 5 – 10 mM TCEP during the solubilization step. (Note however that this must be removed before a standard BCA assay or use a reducing reagent compatible BCA assay.)
With extensive solubilization, it may be useful 1) to sparge your solutions with nitrogen or argon and to keep the protein sample under the same atmosphere (to reduce oxidation) and 2) to do an initial extraction (remove the supernatant and keep it) followed by a harsh extraction. Sequential extraction can be useful to 1) preserve the chemical integrity of the proteins which more easily go into solution and 2) to provide different classes of proteins. Frequently, the protein of interest is more concentrated in one of the fractions.