Protocol Online logo
Top : Forum Archives: : Transfection and Transduction

co-transfection & transfection - (Jun/03/2007 )

smile.gif hello,

when the disired gene transfected in the PC 12 cell, we can use several vectors such as pcDNA 3.1 .

IN my case, I am going to contruct using pEGEP vector.

when I read papers related this project, I seached three method.

FIRST, normal vector + pEGFP

SECOND, constructed pEGFP

THIRD, normal vector -> immunohistrochemistry

I roughly UNDERSTRAD about these method.

give me some information of these method's merit and demerit, please~~^^*

-shi21ne-

I am not sure that I get it right, but roughly you want to express your protein in PC12 cells and you are debating if you are going to recognize the transfected cells by either 1) co-transfection with pEGFP or 2) transfect fusion EGFP protein (i.e. your protein is tagged with EGFP) or 3) IF

If I got your meaning right, then:
- Method 1: desired protein will be expressed as natural proteins (as in, they won't have tags). That is good since the protein's functions won't be interfered by tag. Normally transfection ratio in that case will be with much less EGFP indicator, so that cells express GFP will pressumably also got your protein. However, if you don't have antibody against your protein, you won't be able to detect it, so localization, IP, IF, WB... directly against your protein is out. You can see effects, though, such as see if anything happens to other known proteins or cell structures (which you have Abs) in cells overexpressed your protein.
- Method 2: if the construct is fine, then GFP signal will be your proteins signal. You can do all experiments that require the detection of your protein even if you don't have Ab for it by using the GFP tag. But be careful to make sure that this tag won't affect your protein's funtions.
- Method 3: Again, protein expressed won't have tag, that is good. But this method, unlike the 1st, only work if you have specific Ab for your protein

-Almasy-

You could also add a small tag to your gene in the original vector and transfect it and there is the antibody against the tag if you dont have a good antibody for the protein.

Method 2: Having GFP to the protien might not be the best as it may completely change the way the protien behaves.

What exactly do you want to do? Depending on this, you should select between 1 and 3. But in the mean time, also go ahead with method 2 and verify if GFP makes the protien behave differently and compare it to methods 1 or 3. One can learn a lot of things by comparing them.

Good Luck !!!

-scolix-

thank you for your answer.^^* biggrin.gif





QUOTE (scolix @ Jun 5 2007, 12:53 PM)
You could also add a small tag to your gene in the original vector and transfect it and there is the antibody against the tag if you dont have a good antibody for the protein.

Method 2: Having GFP to the protien might not be the best as it may completely change the way the protien behaves.

What exactly do you want to do? Depending on this, you should select between 1 and 3. But in the mean time, also go ahead with method 2 and verify if GFP makes the protien behave differently and compare it to methods 1 or 3. One can learn a lot of things by comparing them.

Good Luck !!!

-shi21ne-