Dear Researchers,
I cloned the IRF9 coding sequence into plasmid LV073.
I used the Gibson assebly (NEBuilder) protocol.
I cut the vector in MCS with the enzyme ScaI (blunt ends), amplified my coding sequence with cDNA homology arms (Takara HF polymerase), then ligated (NEBuilder), transformed etc.
I have received many colonies. I chose a few of them, performed a miniprep, and transfected the 2fTGH cell line with Lipofectamine3000. After 48h, I harvested the cells, isolated the protein (RIPA + inhibitors) and performed gel electrophoresis (Bis-Tris 4-12%), semi-dry transfer and WB.
I have sequenced the plasmids and the insert looks fine. No indels or mutations.
But on the WB I observed three bands. Only in cells transfected with the IRF9 plasmid. One shorter and one longer. The appropriate size of IRF9 is ~ 48kDa.
What happened?
I am enclosing a photo of WB and a map of LV073 plasmid.
Thank you for the advice :)
Some antibodies are not perfectly specific and may cross-react with other proteins in the extract, leading to additional bands.
Bands of lower-than-expected molecular weight could be due to proteolysis of the protein.
Bands of higher-than-expected molecular weight could be due to post-translational modification, especially glycosylation, or to read-through of the stop codon, or to failure to process an N-terminal leader peptide completely.
I quickly checked few blot images of the protein and found similar bands with IR9. And also uniprot has documented few isoforms. Can you provide antibody details of ur protein.
Hi, Krishna kumar Ganta !
I used two antibodies and results were similar.
Santa Cruz IRF9 mouse monoclonal (H-10)
Cell Signalling Technology D2T8M rabbit mAB
I have never seen these extra bands when checking endogenous IRF9.
Adam B Shapiro thank you for the answer!
Do you think that visible lower band (after proteolysis) is a result of eleveated amount of IRF9 protein in the cell? Why it is not visible in case of endogenous protein? After IFN treatment the amountof IRF9 is quite high, but I've never observed more bands on WB.
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