Best method to locate a receptor on breast cancer cell? - Using an AFM (Jul/16/2008 )
I have access to an AFM (Atomic Force Microscope), which is sort of old, and is only able to view in a dry environment, therefore any cells that we view under the microscope must be fixed (dead.)
Anyways, i am trying to see if viewing receptors on cells through the AFM is possible, and if so how/what method? The AFM can view with both contact and tapping mode.
Also how do you know what a receptor looks like on the topographic map displayed by the Atomic Force Microscope. I know it'll be something with a great hieght, but how can you be certain its a receptor. Also any estimates on what height this should be?
Additionaly, I have started to make bioimprints (take PDMS and pour it on the cells, to preserve the cell surface onto the PDMS model - essentially called a bioimprint). Bioimprints enable us to view live cells (although they aren't really alive because of the bioimprint.) The only downside with the AFM is that it may not show receptors...
Thansk in advance
I'm well familiar with cell receptors (investigating, labeling, purifying, identifying, etc...) and i'm familiar with AFM.
IF you are looking for an easy way to find, or identify receptors on cells I would say AFM is NOT THE WAY TO GO. AT ALL!!!
- Flow cytometry would be the easiest if you want quantitative data
- Confocal microscopy would be the easiest if you want spatial data
If you use an AFM you're looking at some serious limitations
- non-specific binding is significant and difficult to overcome
- you have to coat the micro-cantilever with either a ligand for the receptor or an antibody for the receptor, when you do this you will NOT have control over the orientation of the protein.
- you have to have a VERY WELL cultured MONOLAYER of cells that grow on a GLASS COVER-SLIP... that in itself presents problems and changes to the morphology of the cell...
I realize I do not know all of your circumstances and details, but in my humble opinion and as one who is familiar with receptor biochemistry and AFM I seriously recommend you not use AFM for this purpose.
Yea .. i figured that AFM was not the way to go ... but the thing is I have to. The research that i'm doing is very vast, and a small portion of it is to see if a receptor can be detected by an AFM (and only an AFM). My hypothesis is yes they can be ... the only thing is how should I go about. When you that by coating the antibody/ligand I will not have control over the the orientation of the protein ... what exactly do you mean? One more thing ... the cell culture is growing on a glass flask ... what's wrong with that (I can’t fully comprehend the third bullet.)
Also a quick question: Is confocal microscopy the same thing as fluorecense microscopy?