Co-Transfection for Immunoprecipitation - (Jun/30/2008 )
I'm going to immunoprecipitate 2 gene products with different tags by overexpressing both genes in cells (I use HEK293T).
Therefore, I have to co-transfect 2 plasmids into cells. However, I found they are expressing in different efficiency, one of the constructs is not expressing so well and it seems to me that co-transfecting another construct will inhibit the expression of the low expressing one.
Is this normal?
Some experimental details:
I transfected 10-15µg of the low-expressing one and 3-5 µg of the well-expressing plasmid into 100mm plate.
Calcium phosphate transfection was used and the duration was ranging from 16 hours to 20 hours.
Therefore, I have to co-transfect 2 plasmids into cells. However, I found they are expressing in different efficiency, one of the constructs is not expressing so well and it seems to me that co-transfecting another construct will inhibit the expression of the low expressing one.
Is this normal?
Some experimental details:
I transfected 10-15µg of the low-expressing one and 3-5 µg of the well-expressing plasmid into 100mm plate.
Calcium phosphate transfection was used and the duration was ranging from 16 hours to 20 hours.
Why should you care about difference in expression?
Even with low expression, it should be sufficient for co-IP, as long as I get the mutual IP to work, I wouldn't care about the expression difference.
Usually the band pattern of "Input" has to be shown. And sometimes from the input, I cannot detect (or can hardly detect) the low expressing species.
The second thing is when I cannot pull down one of them, I can trace whether it is not expressed (or not enough), or due to other reason.
Anyway, thank you your comments.
The second thing is when I cannot pull down one of them, I can trace whether it is not expressed (or not enough), or due to other reason.
Anyway, thank you your comments.
Sure, if you can't see it in input, that is very low expression indeed. You need to be concerned!
1. Change the cell line you are expressing your proteins.
2. Change the promoter of your low-expression vector.
3. Make sure your Antibody is good, by doing some controls, otherwise try a new one.
4. SOmetimes, it works to express two proteins in two cells, mix teh lysate, and do co-IP overnight. It doesn't always work, so not the best bet, but if one protein is somehow decreasing teh levels of other, this can be tried out as a last resort.
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