GST tag - expression with GST (Mar/17/2005 )

Hi everybody,
have troubles ... am expressing 50 kDa protein using GST tag(26kDa) and after purification (using Glutathion Sepharose + reduced Glutathion) my protein is 60 kDa (SDS-gel) Really don´t understand it.
Has somebody any idea what could happened or why?
Thanx + Good Luck!

Hi Ell,

I'm having a similar problem as you did earlier. Did you ever figure it out and if so, could you share your experience?

(my fusion protein is 50 Kda, we get the major band at 60 with also a smaller band at 50kda. Bywestern, only the 50 kda is reactive against a monoclonal antibody against the protein of interest. however, anti-GST picks up multiple bands inlcuding the 60, 50 and probably ~100 kDa)

Thanks

Hey,

Often what you find is that the larger sized bands are simply attributed to glycosylation of a protein. For example one cell surface protein is only 120kda in size but due to glycosylation its can be up 160-200kda depending on the tissue. So look for a deglycosylating protocol and treat your protein to it before running a gel.

Dr Washo

Hi Dr. Washo,

That is an interesting possibility. What are some deglycosylation protocols one can use to test if it is true?

Secondly, my protein is a nuclear protein and thus far, I don't know of any glycosylation sites....

Any other thoughts?

Thanks for your help.

Yo,

Well I just checked the protocol search and this link sounds pretty cool:

Glycotech

Another forum talked about the same things here:

Forum


And here is a paper where they deglycosylated a membrane receptor:

Research Paper Using Deglycosylation

And here is a link to a kit that might help you out (but whether this question is serious enough to justify the investment of money is a different story!):

Kit

I think also you mentioned something important, just double check to see if your protein has any glycosylation sites. Here is one more link describing an investigation into nuclear proteins that are glycosylated, you'll find it an interesting read:

High Mobility Groups

Hope those links help!


Dr Washo

Oh forgot to mention, as a far as the western story is concerned that is bizarre! I'll ask my supervisor about that one and get back to you.

Dr Washo

hi all,
i am having a problem i am trying to express human protein of 55 kDa with GST tag. when i am incubating lysate with GST beads fusion protein is not binding to beads. any suggestions are welcome. thanks in advance

Hi Dr. Washko and everyone,

maybe I explained it bad,I´ll try it again:-)
When I expressed it, it´s as large as asked, but after purification something happend and...protein was smaller.

In detail: I have express 76 kDa fussion protein (50 kDa is protein and 26 GST tag approximatly) and after purification (and without cutting!) I´v got 60 kDa "fussion" protein.

Maybe I can sequenate it, but still am interested what´s with it.
good luck all,

Ell

I use GST as tag in purification too. The binding related to different proteins. In my lab, some proteins were easy in binding, some were dificult.
And, what method you are using to broke cell? If you use sonication, do not oversonicate.

Hello, Shawn,
I use sonication. How long do you sonicate? (how long is it "oversonicate" and how is the effect or manifestation of oversonicating?
Thanx, Ell