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cell Transfection and western blot


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2 replies to this topic

#1 alberto

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Posted 12 February 2004 - 05:45 AM

:( I transfect my human cell line and i see a good efficiency looking the GFP expression but when i want to see the protein that i made express by western, i can't see it. Why? the problem is the antibody, the efficiency of tranfection, the quantity of total protein that i charge on the gel? ...Thanks.

#2 ahockert

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Posted 24 February 2004 - 01:11 PM

Are you transfecting in GFP with your gene of choice, if so are the promoters from the two plasmids the same? I find that if I am transfecting using two plamids with the same type of promoter and I can't get a western signal then the problem is either in the amount of protein I load in my gel, or my antibody. Did you run a positive control lane for your antibody? That should help you determine how much of it you need for your reaction.

#3 il0postino

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Posted 25 February 2004 - 11:59 PM

Come on, you really need to give a little bit more detail about your problem and what you are doing. You prayed for rain and got a draught....... Well there are many sins that could have prevented a blessing.

You should start by crossing out what is not wrong:
Have you ever seen your protein being expressed (by this particular construct (by any other constuct, has anybody overexpressed it before))?
Does the DNA look fine on gel?

Both OK? > then the DNA is probably OK.
What is different between when you did see it and your current experiment?

No,
Then check the DNA carefully. Sequence to check the correct reading frame etc.

Etc.

If you post what you are doing (cotransfection or not etc), what you have found so far (what worked and what didn't or any other info) your prayers will be answered with the help of this forum, i'm sure.

good luck.

PS i'm praying for sun shine instead. ;)




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