[Cytometry] Antibody Labeling Question

Parmentier, Julie Julie.Parmentier at astrazeneca.com
Wed Jul 29 09:18:34 EDT 2009


I just wanted to pass along this follow up to a post a few months back regarding antibody labeling issues a colleague of mine was experiencing....

ORIGINAL POST:

I am trying to label monoclonal antibodies with a green fluorescent dye.  I've tried the Invitrogen AlexaFluor488 kits, both large and small scale, and continue to get antibody precipitation in some of my samples.  The precipitation does not occur upon addition of the bicarb buffer to raise pH, but does begin to occur once the Ab solution is added to the dye.  I have tried changing dye and Ab ratios but this does not seem to help.  In the meantime, I have tried the Lightning-Link Atto488 small scale kit and it gives me labeled Abs with no noticeable precipitation.  The Atto488 works in flow, but does not seem to work for imaging so I would like to continue to try to optimize the Alexa488 protocol, or try another green dye labeling method.

1) Any other suggestions for troubleshooting the Alexa488 labeling to try to minimize precipitation?  Or any other techniques to try to get a green fluorophore on my antibodies?

2) Any ideas on how to calculate degree of labeling with Lightning-Link Atto488 kit?  The formula given in the Alexa488 methods doesn't seem to work for the Atto488, don't know if this is because there is still free dye around in the Atto488 samples.  Tried to contact the company tech support but they haven't responded.  Would like to be able to compare degree of labeling between different antibodies, as well as Atto488 vs. Alexa488.

Thanks so much for any suggestions you might be able to pass on!

FOLLOW UP:

Follow-up to Antibody Labeling question:
I had been experiencing problems with the labeling of an antibody.  When trying to label with small molecule fluorophores the antibody had been precipitating out of solution.  We had tried the Invitrogen kits, all colors of fluorophores (Alexa 488, 568, 596, 647), with the same results.  Changing dye/Ab ratios, incubation times, and omitting the periodic mixing only helped minimally.  We had also tried Lightning Link Atto488 labeling; in that method, we did not see precipitation but our labeled antibody no longer bound properly when tested by flow.

Ultimately what worked for us: labeling with R-PE using the Lightning Link kit (no precipitation observed, and the labeled Ab retained proper binding).  We assume that the charge and/or hydrophobicity of the small molecule fluorophores was interfering with our Ab structure, causing the precipitation.  As PE is a protein fluorophore, it did not affect our Ab's stability in the same way.  To note, this is the first problem of this sort we have encountered, and we think that the problems we had may be related to the specific antibody sequence.  Hope our experience helps if you ever have similar problems in the future.

Thank you to everyone who responded, we greatly appreciate all the feedback we received.

ps -- also to note, we found that to properly calculate the degree of labeling after using the Lightning Link conjugation kits, the labeled Ab must be extensively dialyzed to remove all free fluorophore.  Without dialyzing, free dye + quencher still in the solution causes abnormally high absorbance readings, leading to high DOL calcuations.

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