Protein detection and sensitivity of techniques - (Sep/30/2008 )
I've performed a pulse chase analysis in BHK cells by incubating the cells in the presence of 35S-methionine (20uCi/ml) for one hour.
Could anyone tell me what kind of sensitivity I can expect from the radio isotope? No need for specifics, all I really want to know is would western transfer followed by immunoblotting and ECL detection be more or less sensitive?
Thanks in advance for any info.
Could anyone tell me what kind of sensitivity I can expect from the radio isotope? No need for specifics, all I really want to know is would western transfer followed by immunoblotting and ECL detection be more or less sensitive?
Thanks in advance for any info.
it depends on the SPECIFIC methionine concentration (ratio 35S-methionine/methionine) you use, or do you use only radioactive 35S-methionine without non-radioactive methionine?
I'm kind of confused as to why you are using radioactive isotope if you plan to do a western for your analysis. Typically you would want to put your gel down on whatman, dry it, and then use a phosphoimager screen to detect the radioactivity. If you're planning to do a western, then I would think that a cycloheximde chase (without radioactivity) would be sufficient. Or am I missing something here?
Hmm...maybe I read your question wrong. Are you asking whether radioactivity is more sensitive, or western blotting more sensitive? If so, then it depends on 1. how good your antibody is for western blotting 2. how well your S35 gets incorporated. If you don't have many methionines in your protein, then you might want to get translabel that also has another amino acid labeled. This willl increase your sensitivity. Also you might want to consider lengthening the time of the incorporation (we do 4 hrs). Typically if your antibody isn't very good, then radioactivity will work better.
Sorry that I misunderstood your question at first.
Sorry that I misunderstood your question at first.
The question stems from this; Basically I have a kinase inhibitor present in my medium. My work to date was on western blots an they showed the inhibitor delaying translation of a viral proteins during an infection. The pulse chase was to see if the effect was a universal effect on the cells translation machinery.
Appearence of the viral proteins was seen by western in the presence of inhibitor, albeit at much reduced levels. Pulse-chase did not detect any viral protein translation for the 1h incubation period in the labelled medium.
So the question is asking how sensitive is the radio labelling. Did I not detect viral production because it wasn't there, or just there at too low a level? The western antibody is very sensitive and gives clear specific blots.
Appearence of the viral proteins was seen by western in the presence of inhibitor, albeit at much reduced levels. Pulse-chase did not detect any viral protein translation for the 1h incubation period in the labelled medium.
So the question is asking how sensitive is the radio labelling. Did I not detect viral production because it wasn't there, or just there at too low a level? The western antibody is very sensitive and gives clear specific blots.
Can you give some details of your experiment? Just trying to get a feel for what you're really doing here.