蛋白质表达

Hi guys,

I have a question for you. I made a construct which had been
sequenced. It is in pcDNA3.1(+) and with HA-TAA to stop. The right
size for endogenous protein is 31kD. But when I transfected into COS-7
and run western, the size always is 48kD. The endogenous protein is
round 33kD (invitrogen protein ladder) which looks good.
When I do it with TNT in vitor transcription/translation, it gives me four
bands in western; which are 29, 37, 48 and 70kd. Their density goes
down from the lower to the top. They all have the HA-tag in western.
I will try to sequence the proteins or lable with 35S.

Is there any one know what is happen and could help me to figure?
I sequenced the construct four times and each time it is right and the
"TAA" is in ORF.

What can I do to break down the 3rd or 2nd protein construction?
I harvest my protein with 1%SDS, then boil at 100C for 10min. Add DTT and b-MG in loading buffer and boil again for 5 min. The construct is
921bp. There is no ATG from the end of promotor to the beginning part of construct in the backbone.

Thanks

Greenk