I have cloned ACAP2 in pCMV-HA vector and the frame has been sequence confirmed. I am trying to express in HEla cells by transfecting he plasmid at 2.5µg per well (6-well plate) using Lipofectamine Ltx and plus reagent. but the expression results are not repeatable. once, I got a very good band, few times I got faint band and most of the times nothing. I am harvesting the cells 3 days post transfection and cells are lysed using RIPA lysis buffer (100µl per well) with protease and phosphatase inhibitors.
Hi Sonalika,
I agree with Joydeep to try earlier time points. I would also recommend to co-transfect a control, e.g. a bright fluoresscent protein expression vector to control your transfection efficiency. This can be done by replacing 100 ng of your plasmid or by transfecting a separate well. Always try to plate cells at the same density and distribute them evenly.
Hi,
Have you tried anytime to use the proteasome inhibitor MG132 in your RIPA lysis buffer.
hi
for transient transfection 48h after transfection is good and enough.
you could pEGFP-N1 as positive control in your test for checking of transfection efficiency before run the WB test. transfection efficiency dependent on plasmid purity and concentration (maxi prep 0.5-1 ug/ul OD260/280 = 1.8-2 ) and cells should be in growth phase (happy cells ).
good luck
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