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

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