Protein labeling dyes

[Knecht, David]

We need to label a purified protein with a fluorescent dye for use with a 633 laser. The imaging will be dual with either an mCherry or GFP label. I have seen Alexa, Highlight and Dylight labeling kits from various companies.  Has anyone had experience that would lead to a recommendation for one dye/kit over another?  Thanks- Dave

Dr. David Knecht    

Department of Molecular and Cell Biology

Co-head Flow Cytometry and Confocal Microscopy Facility

U-3125

91 N. Eagleville Rd.

University of Connecticut

Storrs, CT 06269

860-486-2200

860-486-4331 (fax)

[rjpalmer]

We've always gotten decent labeling with the AF kits.  Procedure is
ludicrously simple and fast - you have ready-to-use conjugate in
about 2 hrs.  The stock dye (TFP ester) seems pretty stable - we've
gotten good labeling from a batch over 1 yr old.  Our experience is
based on reactions with ca 0.5 mg of protein.

Have no experience with the other kits.

>We need to label a purified protein with a fluorescent dye for use
>with a 633 laser. The imaging will be dual with either an mCherry or
>GFP label. I have seen Alexa, Highlight and Dylight labeling kits
>from various companies.  Has anyone had experience that would lead
>to a recommendation for one dye/kit over another?  Thanks- Dave
>
>Dr. David Knecht
>Department of Molecular and Cell Biology
>Co-head Flow Cytometry and Confocal Microscopy Facility
>U-3125
>91 N. Eagleville Rd.
>University of Connecticut
>Storrs, CT 06269
>860-486-2200
>860-486-4331 (fax)

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fax [phone]

[Christian-103]

A plant mitochondrial lab here would like to look at the redox state of some living Arabidopsis cells.  After checking the lit, they were able to get a hold of both roGFP1 and 2, which then lands on my desk.

The problem that I have is that the ratiometric intesities are not based on wavelength emission changes, but excitation changes which influence signal strength.  The two preferred excitation lines are 405 and 488 with detection in the normal GFP range.  All of the publications that I have looked at have used the 510 Meta in line switching mode.  I suspect this mode, having never used it, allows differential excitation with same channel acquisition.  I need to duplicate this with a FluoView 500, and of course things are moving in those cells, so it must be quick.  I am concerned with effecting the results with bleaching if I try a z-series with both laser lines.  And I know I can not save the images fast enough to catch the same mitochondria in a frame.

Does anyone have any ideas?

Finally, has anyone tried this type of imaging on a Nikon A1? 

Thanks.

Christian

[Tim Feinstein-2]

Dear Christian, 

As far as I can tell from the extant literature your FV500 should measure roGFP as well as any contemporary confocal.  Sequential line scans in a rasterizing detector such as the FluoView alternate on the order of 0.5 millisecond to milliseconds, so structures as large as a mitochondrion won't have time to move.  Take a look at Gutscher et al. (J. Biol. Chem. 284:31532), who used a Perkin Elmer spinning disc.  I don't think that a spectral detector such as the 510 Meta or the Nikon A1s, which my lab uses, will confer much advantage for this assay.  

All the best, 

Tim

Timothy Feinstein, PhD

Dept. of Pharmacology, University of Pittsburgh

On Dec 3, 2009, at 2:17 PM, Christian wrote:

A plant mitochondrial lab here would like to look at the redox state of some living Arabidopsis cells.  After checking the lit, they were able to get a hold of both roGFP1 and 2, which then lands on my desk.

The problem that I have is that the ratiometric intesities are not based on wavelength emission changes, but excitation changes which influence signal strength.  The two preferred excitation lines are 405 and 488 with detection in the normal GFP range.  All of the publications that I have looked at have used the 510 Meta in line switching mode.  I suspect this mode, having never used it, allows differential excitation with same channel acquisition.  I need to duplicate this with a FluoView 500, and of course things are moving in those cells, so it must be quick.  I am concerned with effecting the results with bleaching if I try a z-series with both laser lines.  And I know I can not save the images fast enough to catch the same mitochondria in a frame.

Does anyone have any ideas?

Finally, has anyone tried this type of imaging on a Nikon A1? 

Thanks.

Christian