Proper western blotting
Posted 16 March 2013 - 10:57 AM
Posted 16 March 2013 - 12:33 PM
I agree with you regarding densitometry, it is pretty dodgy, though the signal from the newer fluorescent antibodies is much much more linear, and hence better for densitometry, than that of HRP/alkaline phosphatase.
How do you mean conclusive? it depends on the experiment - are you looking to see if there is a band (i.e. presence/absence)?, are you doing IP/co-IP? Dose response?
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Posted 17 March 2013 - 10:06 AM
Also, is there a link you have describes these new "linear" antibodies? I'm curious. AFIK, if one is going to do densiometry readings, then a standard curve is needed on your gel. Do the new ABs no longer require this?
Posted 17 March 2013 - 10:25 AM
Posted 17 March 2013 - 11:02 AM
I guess for some proteins it's really difficult to obtain a positive control (or even a negative control)... I use AB's in other applications than WB though (flow cytometry, immunofluorescence etc.) and I really had and have trouble finding cells etc. that do express the proteins I work with for sure !
Do you have trouble because of the ABs? Or because of the protein? How do you make sure your ABs really work and really do bind to the protein of interest? Just take the vendor's word for it? What should one do to validate their Abs? SiRNA experiments if one can not purchase the protein outright to do a positive control on a gel?
Edited by Ubiquitous, 17 March 2013 - 11:03 AM.
Posted 17 March 2013 - 01:10 PM
As for your question on what you can really do to validate your AB, I'm curious to hear what other people here think about it.
Posted 17 March 2013 - 01:15 PM