I was bug by some protein expression problems for quite a long time.
I was working on an scaffold protein called AKAP-Lbc/AKAP13(~300kDa) and I tried to expression the pEGFP-N/Flag tagged AKAP-Lbc (~8kb insert size) into HEK293 cells.(It was a kindly gift from the other lab). When I loaded the protein lysate (in RIPA buffer) containing full-length AKAP-Lbc on 8% western mini-gel and probed by 1:5000 anti-Flag(M2), whole lane was completely darken with thick smear (esp. below ~100kDa). No AKAP-Lbc band observed at ~300kDa region. But this smear didn't observe when the same blot was probed by 1:3000 mouse anti-HA.
But dark smear lanes don't appear neither in truncated version of AKAP-Lbc (crop into several 500aa fragments) or in AKAP79/S-AKAP84. Although expressing some of the truncated parts would result in a bit "dirty" lane (not as much as that of full-length, please see their data in attached pic in planel A), one could still distinguish between specific band at desired protein size and the non-specific bands.
So I read all the papers about AKAP-Lbc, their western blots also looked a bit "dirty". Just the smear is not as darken as I got! I wonder if there is any trick to due with this protein ??
Actually, I had followed strictly what the protocols and instructions from the paper:
(1) HEK293 was cultured in DMEM medium ( I tried both DMEM and MEM medium with 10% FBS, it doesn't help)
(2) Transfect 16-24ug of huge 8kb full-length cDNA into 80% confluenced HEK293 on 100mm plate; 6-9ug for truncated fragments. Then grow 24-72hr in 10% FBS/DMEM to express the protein (I tried from 16 to 24ug, 24-72 incubation, didn't help again)
(3) Lysed with 1% Trion-X buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1% (w/v) Triton X-100) or RIPA buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 1% (w/v) NP-40, 0.2% Deoxycholate). Boiled 5 mins in 100'C heat-box after adding the 6X protein dye. (Both didn't help)
(4) To maximize the transfer efficiency, Wet Transfer at constant 25V o/n using either (20% methanol-0.05% SDS-Tris-glycine) or (20% ethanol-Tris-glycine) (don't help the efficieny of transfer for High MW protein. (I could still see some proteins left in the gel by comassive blue stainning after changing 20% ethanol-Tris-glycine to 20% methanol-0.05% SDS-Tris-glycine tarnsfer buffer. But Prons S stained NC membrane showed that there are enough protein on the membrane even there are quite alot of protein strucked in the gel??)
Also do checked the restriction pattern of the DNA sequencing check, making new maxi-preps of this DNA construct by invitrogen column ... etc. All didn't help.
Noted that I transfect the 12ug GFP-AKAPLbc/pEGFP-N and observed it under the microsope. Only 50% of cells give GFP signals but >90% of 3ug transfect pEGFP-N1 cells give green signal. No-GFP-AKAPLbc could be detected when i loaded these lyszed cells on to western mini-gel. LOL
Did anyone experience the same problems in handling high MW scaffold with seems-to-be low transfection effeciency and weired darken of whole western lanes? Any valunable suggestion for solving this issue?
Edited by Jason S LEUNG, 25 November 2010 - 08:22 AM.