Problem with transfection and protein expression - NO DIFFERENCE BETWEEN UNTRANSFECTED AND TRANSFECTED CELLS (Dec/17/2006 )

Hey guys,
I have a problem with transfection and protein expression.
I engineered different plasmids with different inserts each expressing different protein in pcDNA 3.1 vector. I tried to check their expression by transient transfection in two cell lines (HEK 293 and NIH/3T3).
I checked protein expression using western blot from cell lysates as well as media and cell debries (I pricipitated my proteins using TCA), but I found nothing.
I tried to see if the expression is working or not using pcDNA/CAT reporter plasmid but also I got nothing in both cell lines.
I used fugen 6 for transfection.
Then, I tried to check transfection using lucerifase and fugene6 and it worked.
my western is working, I used postive control (I thought it might be my primary Abs for my plasmids not CAT plasmid) but I don't have positive control for CAT to see if primary Abs I'm using for CAT is working.

Now, I don't know what to do!!!!

I will check my plasmids again including CAT plasmid on gel and check 260:280 ratios
Repeat transfection with CAT only in NIH cells using diferent ratios of plasmid and fugene6

I don't know if there is any other reporter gene in pcDNA3.1 that I can use and its easier to detect (e.g b-gal or somthing similar) instead of western.

I think my protocol for transfection is working since lucerifase assay worked.
my protocol for western in fine (positive control worked)
CAT pcDNA I'm using is from the company.

So, guys please if you have any suggestion or question please let me know I need to finish this transfection ASAP.

Thanks

Hi,
I used different ratios for lucerifase assay ( I mean DNA to Fugene6)

2ug DNA with 1:3, 2:3 and 1:6 ratios gave me 27111460, 287086 and 51164528 respectively.

5ug DNA with 1:3, 2:3 and 1:6 ratios gave me 54055530, overload and 48746616 respectively.

So, I decided to use 2ug DNA with 1:6 ratio according to these results.

yeah I checked my constructs by sequencing and restriction enzymes.

I used 6-well plates (if that what you mean) I have to find out what type tomorrow if you mean the brand.

Thanks genehunter,

If you think your primary antibodies are not working; just try running sds-page and stain it with coomassie blue and look for that extra thick band.

The number is decent, if you used 1-5 ul of lysate out of 1 ml total. You probably can use up to 4 ug DNA /well for 6-well plates and DNA/fugene ratio 1:4-1:6 ratio should work well. If you can find an EGFP expression vector that would be helpful for you to see % of transfected cells. Add a fraction of luciferase plasmid (5-10%) to your target plasmid and do co-transfection to cells, 24-48 hrs later do the protein extraction and assay for luciferase would be better control than CAT. I would suggest that you focus on one or two constructs for now and optimize your condition for each step of the experiment first.

The concern with primary antibody could be real. Direct staining of SDS-PAGE with coomassie blue may not be sensitive enough to pick up new bands. Western blot or ELISA would be better way to detect your proteins.

Thank you guys,
Yeah I liked the cotransfection idea very much.
I got pCMV with b-gal today and I seeded my cells and I will do transfection tomorrow with this one and see how it goes.
We think its because of low expression even if the transfection is working as we saw with lucefirase. That's why I wanna use b-gal. I'll do different ratios just to check.
Coomassie didn't give me anything

All sugesstions are welcomed
Thanks

Dear genehunter,
Could you explain the co-transfection idea in more details!!!

Thanks again

As far as I'm aware pcDNA3.1 doesn't have a Kozak translation initiation sequence tho I assume you have already considered this...besides I know people who've just cloned in cDNA with ATG only and it's worked...

This vector expresses neomycin resistance marker, so if you want to check transfection/expression by Western you could do blots for that, Abcam and Upstate both offer anti-neomycin antibodies.

Thanks,
yeah I added kozak sequence to my constructs

I tried CMV with beta gal usig different ratios and it worked and gave me around 40-50% expression with 3T3 cells.
So, I think it is not a problem of transfection too. The only thing remaining is low protein expression.
What do you think guys is the best way to overcome this problem.
I usualy ppt my proteins from media using TCA and use the ppt proteins from media and cell lysate (no ppt) for western!!

any idea
thanks again