How to quantify wild-type and mutant protein? - (Jul/13/2007 )
I am planning to transfect cell with a wild-type (wt) and a mutant (point mutation) protein.
I want to confirm that both proteins are expressed in a same amount using western blotting, but I found it difficult.
I can't use antibody for the protein because it will react to both proteins, and even if I tag each of them with either HA or myc, affinity of antibodies is different so I can't really compare the band of HA and myc and say there's a same amount of wt and mutant expressed in the cell, or can I? 
Does anyone have a good idea?
I want to confirm that both proteins are expressed in a same amount using western blotting, but I found it difficult.
I can't use antibody for the protein because it will react to both proteins, and even if I tag each of them with either HA or myc, affinity of antibodies is different so I can't really compare the band of HA and myc and say there's a same amount of wt and mutant expressed in the cell, or can I?
Does anyone have a good idea?
may be your mutation alters the pI so that you can discriminate the two proteins in 2D GE and subsequent blot analysis
Do you have the same cells transfected with both the WT and mutant proteins?
If you made 2 transfections, do a western and compare the bands. Use some analysis software to do the quantification for you.
You could also use the myc or HA an compare them. Its kind of the same thing.
Thank you for the replies.
>The Bearer
The mutation is R to C and maybe it alters the PI. Nice idea.
>scolix
Yes. I want to transfect same cell with both wt and mutant protein.
You could also use the myc or HA an compare them. Its kind of the same thing.
So there is no or not much difference in affinity between different antibodies (HA, myc)?
Does most referee think that is acceptable?
>The Bearer
The mutation is R to C and maybe it alters the PI. Nice idea.
>scolix
Yes. I want to transfect same cell with both wt and mutant protein.
You could also use the myc or HA an compare them. Its kind of the same thing.
So there is no or not much difference in affinity between different antibodies (HA, myc)?
Does most referee think that is acceptable?
I am sorry, I guess I misunderstood you about the tags. The HA and myc antibodies can have different affinities but if you could use the different controls to show similar protein in both lanes, it can work. You are going to transfect with 2 constructs but with same amounts of DNA. This by itself indicates similar levels of protein. So if you can show by using the 2 different epitope tag antibodies the presence of similar amounts of protein, the reviewers might buy it.
You could also try the using secondary fluorescent antibody for the Western and use the HA and myc antibodies as primaries and quantify the fluorescence, may be it works to differentiate the 2 proteins. Fluorescent markers for western can give you better gradient levels and might help to quantify the fluorescence using a imager.