I am performing a transfection protocol on HEK293 with cDNA 3.1 expressing CD40-Rluc.
After getting stable transfection, I am getting two band in my westernblot for CD40; one on the weight cd40-rluc and the second for cd40 alone. Further, when I reveal with anti R-Luc, I get one band for cd40-Rluc but no mark for the RLuc alone.
RLuc and CD40 have the same molecular weight.
So I was wondering if some had this problem or if someone know if there is any mechanism that lead to the cleavage of the CD40-Rluc complex and since the CD40 is transmenbrane protein the RLuc will be lost by excretion.
Thank you.
How do you lyse your cells for western blot? The cleavage could be prior to lysis in cells as you suggest, or after lysis if you use a non-denaturing mild lysis buffer. Are you also sure there is no alternative start site in your DNA sequence?
If the problem is before cell lysis....are your stable cells clonal and do you see the same cleavage in all your clonal lines, independent of the level of expression? You may find the problem is reduced with lower expression levels, or altering the sequence/linker length between cdc-40 and your R-luc so it is less susceptible to proteolytic cleavage.
If it's after lysis maybe try a denaturing SDS LB or include protease inhibitors...
Best wishes,
Helen
Please, take into consideration that a DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. In the following review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. We exemplify a number of studies, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. Finally, we conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes.
Please see the following manuscript for details:
Stepanenko AA, Heng HH. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat Res. 2017 Jul;773:91-103. doi: 10.1016/j.mrrev.2017.05.002.
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