2 replies to this topic

[soju]

Hi ,
Here I got few questions regarding to the GST fused protein.
My main aim with this protein is to find unknown interacting proteins.
What I did is..
I expressed my protein in E.coli. it got expressed and it bound to Bead. I sediment the bead at 850rpm in 4degree
I washed 5 times with PBS pH 7.3 (chilled)
After this I loaded in gel I am getting my interested protein but along with this am getting non specific proteins. Please do help me to sort out this.
Once after getting the pure protein I want to incubate with whole drosophila embryo proteins and after washing I want run gel and want to send it for micro sequencing. This is my idea,
Here my second question is what extend my approach is going to work I mean micro sequencing one..,..if any body did this plz help me…

[swanny]

soju, on May 21 2009, 07:07 PM, said:

Hi ,
Here I got few questions regarding to the GST fused protein.
My main aim with this protein is to find unknown interacting proteins.
What I did is..
I expressed my protein in E.coli. it got expressed and it bound to Bead. I sediment the bead at 850rpm in 4degree
I washed 5 times with PBS pH 7.3 (chilled)
After this I loaded in gel I am getting my interested protein but along with this am getting non specific proteins. Please do help me to sort out this.
Once after getting the pure protein I want to incubate with whole drosophila embryo proteins and after washing I want run gel and want to send it for micro sequencing. This is my idea,
Here my second question is what extend my approach is going to work I mean micro sequencing one..,..if any body did this plz help me…

have you tried washing with some glycerol, say 10%? That should get rid of most of the non-specific binders. When you load the embryonic lysates, remember to do the same before you elute to stop Drosophila non-specifics binding.

[soju]

swanny, on May 21 2009, 08:24 PM, said:

soju, on May 21 2009, 07:07 PM, said:

Hi ,
Here I got few questions regarding to the GST fused protein.
My main aim with this protein is to find unknown interacting proteins.
What I did is..
I expressed my protein in E.coli. it got expressed and it bound to Bead. I sediment the bead at 850rpm in 4degree
I washed 5 times with PBS pH 7.3 (chilled)
After this I loaded in gel I am getting my interested protein but along with this am getting non specific proteins. Please do help me to sort out this.
Once after getting the pure protein I want to incubate with whole drosophila embryo proteins and after washing I want run gel and want to send it for micro sequencing. This is my idea,
Here my second question is what extend my approach is going to work I mean micro sequencing one..,..if any body did this plz help me…

have you tried washing with some glycerol, say 10%? That should get rid of most of the non-specific binders. When you load the embryonic lysates, remember to do the same before you elute to stop Drosophila non-specifics binding.

thank you..i will do that. i will wash before going to incubate with embryo preotins and adter incubating. will you please do let me  know the incubation time .
thanks