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[eyes]

Hi as I posted some days ago, I try to detect two very small tagged proteins (about 4 and 17 KDa respectively) encoded by a pGL3 control vector that I transfected in HEK293.
I seeded 2X10⁶ into 100-mm dishes and then transfetcted the following day with 10 ug DNA using Superfect (Qiagen). I lysed the cells in 100 ul RIPA buffer then placed 1 hour on ice and performed two thaw-freezing cycles. After that lysates were centrifuged and stored at -80°C. I decided to use only 100 ul RIPA buffer to keep protein concentrated in order to load as much as possible in my minigel (and following a paper in which in a similar experiment cells were resuspended in 200 ul of NP40 buffer).
I loaded about 15% in a NuPAGE® Novex 4-12% Bis-Tris Gel and ran using the MES buffer by Invitrogen. I blotted onto 0,2 um PVDF membrane.
I was almost not able to detect anything using antiFLAG and anti c-myc antibodies, neither in my controls. I then tried to detect B-actin in order to understand what would have happened to my samples and as you can see in the attached picture the signal was really faint (membrane was exposed for 1').
Do you think I used to less lysis buffer and the cells were not lysed enough? Could you please suggest me what should I do to fix such problems? It's really a while that I've been working on this proteins and as you can understand i really frustrating...
Please help me.
Thanks

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