his-tag protein - (Jun/14/2006 )
I have cloned a gene cDNA into pcDNA3.1 and express in Cos7. Now, i have difficulties detecting my recombinant protein using western.
I used 6-well trasient tfx follow by 60ul NP40 lysis. I loaded 15ul into SDS-PAGE but detecting nothing but unspecific band using anti-his.
The sequence is in-frame. tfx efficiency is good.
Could this be due the the low expression level? what amt is normally enough for WB?
Could this be extraction buffer?
Thanks for any input.
nonspecific means what?
does the protein is dimer?
You can try purify the protein by Ni column and detect whether there is purified protein after flow through Ni column.
The expression level is enough for WB!
I guess pcDNA 3.1 also contains myc tag. Try detecting usign myc antibody if u have one.
What sort of extraction buffer r u using to extract the protein as this could also be a reason.
pCDNA3.1 neither contains myc nor His tags. Did you use pCDNA3.1-myc/His? If not, I doubt you can detect your protein using either the tags or doing a His-tag-pull-down
We use pcDNA and it has myc and His tag. One for purification and other for detection. But there r different pcDNA vectors.
I don't want to contradict but if you check this: http://www.invitrogen.com/content/sfs/vectors/pcdna3.pdf you will find that pcdna3 doesn't have myc or his. Maybe it's pcdna3.1myc/his
Dont want to be rude.
As I wrote earlier, there r different pcDNA 3.1 and since cyk used pcDNA 3.1, i suggested that their vector could contain myc tag along with His.
Perfect, scolix: we agree. It is probably a variant o pcdna3.1 containing mys and his. That's what I asked earlier.