DNA in western blot - (Jan/16/2006 )
Hi,
I would like to check for prot expression by WB after transfection of my favourite construct in HEK cells. The idea would be:
- test different conditions for DNA amount/ reagent concentration/ time of expression/ cell confluency..., using one 60mm dish/condition.
- wash the cells, detach, spin down cells, heat (or not?) in SDS sample buffer, minimal amount (ideally less than 50µl: I want to load minigels)
- load gel and blot
Now:
- I tried heating (2 min 90°C), then centifuge 15 min 15000g: the DNA will not pellet
-so I sonicate (10 sec). It breaks down the DNA, but I have a DNA smear on my gel (Ponceau)
- And although the gel looks OK (no distorsion of lanes), I don't see any expression of my transfected protein (I have a positive control for the antibody on the same gel= OK)
So i don't know if my transfection conditions are not good, or if the DNA in the samples messed up the detection. And I would really like to get rid of it, just to be sure.
Thank you for your advices!
I would like to check for prot expression by WB after transfection of my favourite construct in HEK cells. The idea would be:
- test different conditions for DNA amount/ reagent concentration/ time of expression/ cell confluency..., using one 60mm dish/condition.
- wash the cells, detach, spin down cells, heat (or not?) in SDS sample buffer, minimal amount (ideally less than 50µl: I want to load minigels)
- load gel and blot
Now:
- I tried heating (2 min 90°C), then centifuge 15 min 15000g: the DNA will not pellet
-so I sonicate (10 sec). It breaks down the DNA, but I have a DNA smear on my gel (Ponceau)
- And although the gel looks OK (no distorsion of lanes), I don't see any expression of my transfected protein (I have a positive control for the antibody on the same gel= OK)
So i don't know if my transfection conditions are not good, or if the DNA in the samples messed up the detection. And I would really like to get rid of it, just to be sure.
Thank you for your advices!
Sorry but it's not really clear
If you try to lyse cells from a 60mm dishes in 50 microliters I guess it's completely un feasible there's too much DNA in this amount of cells you will end un with a mega chewing gum.
If you look for the expression of your transgene why don't you check the protein itself by western blot ?
Is it a transient transfection or a stable transfection ? Do you tend to think that you will get integration or not ?
As far as I know Ponceau is used for staining Proteins not for DNA why don't you use ethidium bromide to satin your Southern Blot ?
Give us more hints and we will try to give you some help
Pesji
Yes , really I was not clear at all, Pesji! as I said I am not doing a southern but a western blot for detection of my transfected prot .
To rephrase the question: how do you get rid of DNA when you directly lyse cells in SDS sample buffer?
I really dont like just lysing my cells directly in sample buffer as pesji mentioned earlier.....it results in a mega chewing gum like consistency.
The first thinng you need to do is decide how much of protein u want to load...20-40 ug of protein is usually loaded.
The easiest thing for you to do is pellet the cells and sonicate in a Ripa or Tris buffer. Then you spin the lysate and do a protein reading on the suspension. Combine the appropriate amount of suspension in lemli buffer .....boil and load. This is the easiest way to get rid of DNA in the lysate and also will solve the sticky problem....Good luck.
Let me know if u need the recipie for the ripa buffer....
p..
In a previous lab we used to make lysates for Western blot directly in gel sample buffer (1,000,000 cells per 100µl GSB), then sonicate for 30secs or so (the tube should heat up slightly), then boil for 5mins on a heating block and load up to 10-20µl. You shouldn't need to pellet anything after sonication, but the sonication should reduce the gloopiness by shearing the DNA and make the sample easier to load.
Is your positive control purified protein or a control cell lysate ? If you're using pure protein it might not be a fair comparison if your protein is expressed at low levels. A cell lysate might be better and load as much sample as possible. Since you're using 293 cells I'm guessing your transfection efficiency should be high but it might be worth checking with GFP down the FACS or microscope. Try a someone else's plasmid from which they know they can detect expression to check the system is working. I'm guessing you're using a strong promoter (e.g CMV) so you should also get high expression levels unless the protein is toxic etc.
Good luck,
Ceri