Confirmation of protein expression in pCDNA3 - How to know foreign gene expression in pCDNA3 (Mar/12/2008 )

Dear All,
I am in a very big problem and need some suggestions. I had my gene cloned in pCDNA3. I want to know the best way to confirm its expression. Can you please name few of the best and easiest methods available (money is not the constraint)?
Thanks a lot in advance.
Waiting eagerly...
Niraj

---

Do a transfection in any cell line you have using any transfcetion method. Then, collect your media if you think your protein will be secreted or lyse your cells and do a western to check your protein expression. It takes 5 days to finish if you have everything ready.
If you have GFP you can look under the microscope for GFP expression without doing WB.

Let me know if you need more info
Good luck

---

ya I think the easy and best way is to do western with cell lysate or the media( if secreted) after transfecting the cells. be sure to put control i.e untransfected cell as the cell previously may contain the protein as endogenous.

---

Thanks a lot to both of u.
Can I use 293T cells for transfecting the pcDNA3+my gene and pcDNA3 as control?
Hope to hear you soon.
Niraj

---

yes you can. But I would use pcDNA expressing a reporter gene too like beta gal or GFP easier for optimization.
Your empty pcDNA is your negative control.

Good luck

---

So, I think I can have experiment with three constructs
1) pcDNA3+my gene
2) pcDNA3+reporter gene
3) pcDNA3
Thanks a lot for quick reply.

---

This plan is fine. Hopefully, you have antibodies to detect expression of protien on western.

good luck !!!

---

Thanks a lot!
Yes, I have the antibodies against the protein of interest. I can check the transfection by looking at fluorescence of the GFP. Right?

---

Yes!

---