Hi all,

I am not experienced with the Phos-Tag Acrilamide system but, from what I am seeing I am doing something wrong.

I do my gels in Bis-Tris (NuPage-type), MOPS for the electrode buffer and use LiDS-sample buffer (home-made but similar to Thermo's) for loading. Strictly no EDTA in all cases. My samples (yeast total protein) are prepared in RIPA buffer with phosphatase inhibitors (vanadate and beta phosphoglycerol) and EDTA; however, knowing that this can cause problems, I precipitate my samples with TCA or Cl3CH/MetOH prior to resuspend in LiDS-sample buffer.

I run 7.5% acrilamide gels with 25 microM Phos-Tag, 50 microM ZnCl2 at 15 mA, constant current, and stop when the Coomasie front reaches the end of the gel. After transfer to nitrocelullose and staining with PonceauS, I consistently see all my proteins in the top half of the gel, nearly nothing in the lower half, and bands are distorted (M-shaped).

Heat is, I believe, not an issue (buffer is not hot after run and I keep my gels "immersed" to help heat dissipation). When I do my gels without Phos-Tag, they run beautifully.

I include an image of the last PonceuS I did just today.

Any ideas of what could be the problem here?

Thanks a bunch