I have already try to dry the sample about 20 minutes and to dissolve in miliQ water and do not work too.

What do you mean with "dialyze" the sample? Just left the sample RT 3h or overnight in miliQ with beta-mercapto?

If there is so much protein in the TCA pellet that it won't dissolve in Laemmli sample buffer, you probably didn't need to use TCA precipitation in the first place. If you still have some of the original sperm nucleus sample, try dissolving it directly in the sample buffer, skipping the TCA precipitation.

The sample can be easily dissolve before TCA and acetone, but I believe it could be the sperm nucleus (I'm using 20 millions sperm/sample). I aready tried to quantify the protein using Qubit assay, and the concentration is too small, around 12 to 15 ug/samples, but this quantification was performed with a sample that I could't dissolve. I gess the most part of proteins remaind unsolved and that's why I have this small concentration.

Hi,

1- I washed the TCA precipitate once with Ice-Cold Acetone containing 0.1% HCl, vortex, and centrifuge at 17000 xg and 4C.

2- I washed it once with Ice-Cold Acetone and centrifuged it at the same conditions.

3- The washed protein precipitate was air-dried.

4- The dried pellet was dissolved in lysis buffer (10% glycerol, 2% SDS in 62.5 mM Tris-HCl pH 6.8), vortex, and sonicated for a total of 70 sec (10s per cycle, pause for 30 sec between each cycle to avoid the heat).

5- Centrifuge at, 17000 xg and 4C.

6- Transfer the clear supernatant to a pre-chilled tube and determine the protein concentration.

7- If you are going to perform SDS-PAGE and western blot, just add beta-mercaptoethanol (710 mM, ex. add 5 uL Mercapto for 100 uL of the lysate) and the bromophenol blue (0.005%)

8- Boil for 10 min at 100C.