cell fractionation and microscopy - (Oct/12/2014 )

Hi

Has anyone in their experience seen that results obtained from subcellular fractionation completely match those obtained by doing fluorescence/confocal microscopy? I am answering reviewers on the data that I have by both techniques where the protein I am studying looks much more nuclear by confocal and fluorescence microscopy than it does by looking at its distribution in the cytoplasm and the nucleus by fractionation.

Thanks

Swati Dhar

The results should match. What controls are you using for your fractionation? Are you using the same antibody for IF and western and at the same concentration?

Thanks bob1. I have Lamin A as nuclear control and Erk1/2 as cytoplasmic control in cell fractionation. Both are pretty neat and show no cross contamination. However, the antibody that I am using for IF or confocal is different from the one I use for regular WB and the antibody is rabbit polyclonal for IF/confocal as opposed to rabbit monoclonal for WB. For IF, I have single antibody stained controls and they show pretty much the same as the dual staining. One of the proteins is entirely nuclear which is known while the other which I am interested in is supposed to be majorly cytoplasmic reflected by fractionation results and some distribution in the nucleus also as seen by fractionation but not in confocal/IF. The latter technique is showing a little more nuclear which is hard to interpret. However, I have seen number of papers which do show confocal/IF images for the same protein much the same way as in my results. As far as I know, for most proetins it is difficult to use the same concentration for an antibody both for IF/confocal and WB.

Ok, those controls sound fine. It may pay to over-expose your control blots just to see if there is some faint contamination coming through. If your the antibodies against your protein of interest are more sensitive than the ones used for controls, it might be that you are getting some cross contamination which isn't visible on the control blots.

One of the difficulties with doing IF/ICC/IHC is how do you know that the pattern you see is specific? If you have an antibody and use it for WB at one concentration (say 1:1000 for discussion purposes) that gives you a specific band (and no other bands), but gives multiple banding at higher concentrations (e.g. 1:500, 1:250) and you then use those concentrations for IF... what is the contribution of those non-specific bands to the cellular localization you see?