How does that compare to your expectation? On a related note, is the overall accuracy of quantitation higher with Li-COR/FL than blot-strip-blot with CL? Many thanks!
As far as I know there is no significant interference neither in theory nor practically betwenn 680 and 800 channels.
It's a great system since you may incubate up to 4 antibodies simultanously if species and size of the detected bands differ. The quantification range is much more accurate than CL.
The advantage of CL developed by film or camera is that the signal accumulates and you may detect very little abundant proteins much better by a long-time exposure by CL than by LICOR.
I mixed HRP and fluorescent secondary antibodies successfully incubating simultaneously and the detecting first the fluorescence and then CL by ECL/Film.
Good luck!
Thanks Stephan! My particular concern deals with quantifying phosphorylation in which case the phosphorylated protein has very similar MW as the non-phosphorylated,. My abs work fine individually, and I am trying to figure out the best way to quantify the % of phosphorylation which sometimes can be low.
Completely agree with your comment on CL, precisely why I look elsewhere for quantitation. what's your tolerance for interference between channels, percentage wise?
appreciate the help!
Have a look on Licor and Cell Signaling Tech. homepages under the term in-cell western and in-cell western validated antibodies (from Cell signaling), which are exactly what you may be looking for. See if your Kinase is validated!
http://www.cellsignal.com/common/content/content.jsp?id=in-cell-western
http://www.cellsignal.com/common/content/content.jsp?id=ourApproach-validation-if
ST
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