Do you have protease and phosphatase inhibitors added to your lysis buffer? Skin has a lot of metalloproteinase activity. Also, how much protein you have per sample? Even coomasie assay has the limits of detectiion.

I totally agree with Dr. Edita's answer, attached protocol that could help. It is important to use protease inhibitors and ask if the western blot is done only under denaturing conditions (SDS) or if you do it under denaturing and reducing conditions (SDS and a beta-mercaptoethanol type reducing agent). The gel could visualize it with staining with silver.

Preparation of whole cell lysate

Collected cultured skin cells were suspended in 150 μl of PBS containing a proteinase inhibitor cocktail (Calbiochem, La Jolla, CA). Cells were sonicated on ice for two strokes (10 sec per stroke) using a Fisher Sonic Dismembrator (Model 100, Scale 2). After incubating on ice for 30 min, cell lysate was centrifuged at 14,000 x g for 15 min, and the supernatant was collected and designated as Whole Cell Lysate.

Western blot analysis

Whole Cell Lysate was used for the assay. Antibodies against ATF2 (sc-187), Fra-1 (sc-605), α-Tubulin (sc-5286), and β-Actin (sc-47778) were purchased from Santa Cruz Biotechnology.

Exactly José Ramón Vielma ·

Thanks Edita,

For the point of using Protease inhibitor.

But I always use 1X protease inhibitor cocktail (sigma). Still I failed to see. 

And Thanks to Jose.

However, my sample is skin epidermis directly from mice after cryofrozen in Liq-N2.

So while homogenizing and after sonication/centrifuge I see huge pellet. (Not like while you lyse culture shin cells). I will try to keep the sample on ice for 30 min as you recommend.

Thanks.