Dear Aabid Hussain Ph.D

it depends from the cell lines that you would like to use.

in my experience the pcdna3.4 and pcdna3.1 wotk quite well with HEK293, EXpi293 andCHO. Whenyou are desing your cloning you need to add to your forward primer the Kozac sequence, the starting ATG codon, an N-terminal signal peptide to acheive protein secretion as well as ae stop codon in the reverse one.

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good luck

Manuele

Dear Dr Manuele Martinelli

Thank you so much for your help.

We used the Expi293 cell line, and our insert (X) was cloned into the pcDNA3.1(+) vector. The final construct is pcDNA3.1(+)-X.

However, we haven't observed a prominent band on the Coomassie-stained gel. Our goal is to detect a clear band using Coomassie staining.

If you have any further suggestions, they would be greatly appreciated.

Best regards, Aabid

Did you add at the N-terminal the KOZAC sequence and the leader peptide for protein secretion?

How big is your protein? And it is codify for a souble domain of your domain contain some transmembrane regions?

Take in consideration that expecially for high mw protiens, the protein in the media could be quite diluted, could be normal that you do not we haven't observed a prominent band on the Coomassie-stained gel, since the media represent an high volume respect to the cells and the protein is quite diluted.

In many cases to detect it you have to run a WB instead SDS-page or otherwise run a small scale purification to enrich it:

Generally with Expi293,when i have the his tag, I perform a rapid small scale IMAC purification loading 1-2ml of surnatant in 100ul of Ni-NTA resin and eluting with max 300ul.

Details in the following link:;

https://proteocool.blogspot.com/2021/05/tips-of-week7small-scale-imac.html

in this way you will enanche the detection of your SDS-page

good luck

Manuele