By which method you have reached an ideal/clearer staining?
Hi Hercules,
As Anand suggested, there really is no best protocol as it is entirely dependent on your epitope and the method of fixation. Most people use either 3% Paraformaldehyde (PFA) (typically 30' at room temp.) or 100% MeOH (typically 7' at -20 degrees).
PFA will cross link your protein of interest and fix it's 3 dimensional conformation. As such your antibody will need to recognized an exposed loop or a 3 dimensional epitope. Furthermore, PFA will not permeabilize your cell. Thus you will need to do so. I use 0.2% Triton X in PBS for 5 minutes. There are several other detergents you can use as well. MeOH on the other hand, will permeabilize cells and slightly denature proteins. As such, it can either reveal or destroy epitopes.
Typically, when I am testing a new antibody, I'll try both to see which works best and compare the signal to what is known about your protein of interests. If you have knockout/knockdown cells, even better. Then you can verify that your signal is specific. It is also important to note that if you see different staining patterns with different fixations, it may not mean that one is wrong. It may mean that you are seeing different pools of your protein within the cell.
Happy to send specific protocols if you are interested, though you can find many online.
Good luck!
Hi...
Depends on your protein (its localization) & quality of antibody.
Standard protocol is
1) Saponin treatment - widely works - mild treatment to the cells
2) Triton (0.1 % to 1% ) - works well for cytosolic proteins - good for some proteins
3) Methanol (cold fixation) - works well for several proteins (not recommended for GFP / RFP exressing cells, signals is killed by methanol)
Try & standardize best for your cells - antibodies - structure of interest
All protocols are available online..pretty standard !
Good luck
Anand
Hi Hercules,
As Anand suggested, there really is no best protocol as it is entirely dependent on your epitope and the method of fixation. Most people use either 3% Paraformaldehyde (PFA) (typically 30' at room temp.) or 100% MeOH (typically 7' at -20 degrees).
PFA will cross link your protein of interest and fix it's 3 dimensional conformation. As such your antibody will need to recognized an exposed loop or a 3 dimensional epitope. Furthermore, PFA will not permeabilize your cell. Thus you will need to do so. I use 0.2% Triton X in PBS for 5 minutes. There are several other detergents you can use as well. MeOH on the other hand, will permeabilize cells and slightly denature proteins. As such, it can either reveal or destroy epitopes.
Typically, when I am testing a new antibody, I'll try both to see which works best and compare the signal to what is known about your protein of interests. If you have knockout/knockdown cells, even better. Then you can verify that your signal is specific. It is also important to note that if you see different staining patterns with different fixations, it may not mean that one is wrong. It may mean that you are seeing different pools of your protein within the cell.
Happy to send specific protocols if you are interested, though you can find many online.
Good luck!
Dear colleagues,
Thank you for the thoughtful comments, I'm comparing some methods others use to improve mine. Appreciate any more comments and experiences with the method.
Sincerely,
Hércules R. Freitas
Michael,
I'm currently working with purinergic and pyrimidergic receptor signalling, especially in glial cells.
Sincerely,
Hércules Freitas
There can be multiple factors, which I'm not going to go into here (I've given hour-long webinars on it and still just skimmed the surface). I recommend you contact us at Molecular Probes Tech Support and we can guide you (feel free to ask for me). 541-335-0353, or 800-955-6288, then option 4, then 3, then 2 in the phone tree.
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