We are using expression vector pcDNA3 HA Arf6 ActQ67L (Plasmid #10835) obtained from Addgene. But we could not detect the expression of the product through WB using an anti-HA antibody (both clone F7/Y11).
Please tell me how to troubleshoot. Or should we use another plasmid that will express Arf6 Q67L tagged with HA or FLAG?
The transfection procedure was as below,
Cell: Cos7 (cultured in 24 well plate by 1ml medium)
Transfection reagent:X-treme GENE HP DNA transfection reagent
Plasmid:Arf6-Q67L
1) Add 100μl opti-mem and 1μg plasmid
2) Vortex for few seconds and leave to stand for a few minutes.
3) Add 2μl X-treme
4) Vortex for few seconds and leave to stand for 20 min in room temperature
5) Add to cultured cells drop by drop and incubate for 6hr
6) Change medium to the regular medium
7) Incubate for 18hr
Did you include a positive CTL in your WB? Did you check that the antibody is working properly?
Tetsuya Takahashi
Like Sebastian mentioned. When working with transfections, you always need a GFP (or any fluorescent protein) positive control, to make sure that the transfection protocol is working. If your positive control has GFP expression, your non-GFP plasmid (in a separate transfection) should be working. So that would point you into the western blotting. If there was no GFP than you trouble shooting transfection. For your WB did you run a positive control? Like an Actin or Gapdh? With a new plasmid, I would have it sequenced and I would transfect with a larger amount of cells until you have established the transfection protocol working properly before switching to smaller format transfections.
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