I am working on AKT phosphor and tot protein. I have blots with both proteins and need to quantify the phophoAKT band relatively to the total AKT band, both bands being detected by 2 different Ab. When acquiring the signal (using a chemi geldoc system and a CCD camera, Alliance system) the pAKT band has a different threshold of saturation than the total AKT. For instance, if the saturation of pAKT band starts from 1.5sec of exposure, the total AKT band would start getting saturated at 0.5s. My question is this one: when doing the ration pAKT/totAKT, which exposure time should I pick for each band? should it be pAKT 1.4s/totAKT 0.4s (I picked 0.1s below the overexposure time to avoid using the threshold saturation value)? or should it be the same exposure time for both bands e.g. pAKT0.4s/TotAKT0.4s? I have noticed that depending of the exposure time, the value of the quantification vary.
Thank you for your insights.
Cheers
Like Andreas I would caution quantification of western blots (particularly using two different antibodies) by densitometry.
If I were you I would attempt to normalise your lysates based on total AKT levels (much like a housekeeping gene). Then probe for pAKT. Im not sure what others think of this method but it would give you a clearer picture of the different levels of phosphorylation in your samples.
Hi Prica,
we just publish a method to do that (DOI: 10.1038/srep16995). We are able to quantify the amount of protein phosphorylation per cell using this protocol and you can also obtain a percentage of phosphorylation for your protein of interest. You will need the recombinant protein to quantify the total protein present in the samples, and immunoprecipitate one sample to quantify the phosphorylation level. Our method uses a different visualisation system than yours but you should be to adapt it. Best wishes. Francoise
Thanks a lot you all for your valuables input from which I learn new methodology of quantification! @ andreas, I will definitely try the phos-tag gel. @ Francoise, the high density immunoblotting methodology sound easy but yet very clever. Best wishes
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