direct labelling of primary antibodies

[Sylvie Le Guyader]

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Dear list

Directly labelled antibodies are commonly used in FACS. The great advantages in using them are:

  *   No need to incubate with a secondary (expensive, time consuming, large, killing animals)
  *   No trouble finding secondaries raised in different species to avoid cross-binding when one labels with more than 3 antibodies.

Unfortunately for microscopy, one often wants to use an antibody that is not available with direct labelling. There are several companies that offer kits to directly label the primary antibody.

I would like to hear the experience of the list about direct labeling methods.

We have tried Flexistain<http://kromnigon.com/flexistain/> and Mix-n-Stain<https://biotium.com/product/mix-n-stain-cf-dye-antibody-labeling-kit/>.

The first one requires Biotinylated primaries so that is a disadvantage if one already has an primary that works in the lab.

With Mix-n-stain we have had mixed results where the labeling worked nicely with some antibodies and not at all with others.

My questions are :

- what is your experience if any, with the kids mentioned above?

- could you recommend a way to directly label antibodies that works well for you?

Thanks

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader, PhD

Live Cell Imaging Facility Manager

Karolinska Institutet- Bionut Dpt

Hälsovägen 7C,

Room 7362 (lab)/7840 (office)

14157 Huddinge, Sweden

mobile: [phone]

LCI website<https://ki.se/en/bionut/welcome-to-the-lci-facility>

Follow our microscopy blog!<http://microscopykarolinska.se/>





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[Gaurav Joshi]

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Hi Sylvie,

While I have not used the kits mentioned in your message, I use the rabbit IgG conjugation Zenon labeling kit from ThermoFisher.
https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/protein-and-antibody-chemical-labeling/antibody-protein-labeling-kits/zenon-antibody-labeling-kits.html <https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/protein-and-antibody-chemical-labeling/antibody-protein-labeling-kits/zenon-antibody-labeling-kits.html>

- It works exceptionally well and as advertised.
- I conjugate rabbit IgG (primary antibody) with the Zenon conjugation reagent 488. It is used on FFPE sections that are pre-labeled with primary (unconjugated rabbit and unconjugated mouse) and secondary (anti-rabbit 647 and anti-mouse 595) antibodies. This allows me to multiplex 3 antibodies.
- The rabbit IgG conjugated to 488 using zenon does not bind interact with anti-rabbit 647.
- It takes 10 minutes to conjugate and the conjugate is ready to use.
- It’s better if your primary antibody that you intend to conjugate with Zenon is at a higher concentration as the antibody gets diluted by around 10 folds towards the end of conjugation process.

Best,
Gaurav.

Gaurav Joshi, Ph.D.  
Postdoctoral Fellow | Sarosiek Laboratory
John B. Little Center for Radiation Sciences
Harvard T.H. Chan School of Public Health
677 Huntington Ave | Building 2, Rm 229 | Boston, MA 02115

> On Aug 23, 2019, at 1:47 AM, Sylvie Le Guyader <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear list
>
> Directly labelled antibodies are commonly used in FACS. The great advantages in using them are:
>
>  *   No need to incubate with a secondary (expensive, time consuming, large, killing animals)
>  *   No trouble finding secondaries raised in different species to avoid cross-binding when one labels with more than 3 antibodies.
>
> Unfortunately for microscopy, one often wants to use an antibody that is not available with direct labelling. There are several companies that offer kits to directly label the primary antibody.
>
> I would like to hear the experience of the list about direct labeling methods.
>
> We have tried Flexistain<http://kromnigon.com/flexistain/> and Mix-n-Stain<https://biotium.com/product/mix-n-stain-cf-dye-antibody-labeling-kit/>.
>
> The first one requires Biotinylated primaries so that is a disadvantage if one already has an primary that works in the lab.
>
> With Mix-n-stain we have had mixed results where the labeling worked nicely with some antibodies and not at all with others.
>
> My questions are :
>
> - what is your experience if any, with the kids mentioned above?
>
> - could you recommend a way to directly label antibodies that works well for you?
>
> Thanks
>
> Med vänlig hälsning / Best regards
>
> Sylvie
>
> @@@@@@@@@@@@@@@@@@@@@@@@
>
> Sylvie Le Guyader, PhD
>
> Live Cell Imaging Facility Manager
>
> Karolinska Institutet- Bionut Dpt
>
> Hälsovägen 7C,
>
> Room 7362 (lab)/7840 (office)
>
> 14157 Huddinge, Sweden
>
> mobile: [phone]
>
> LCI website<https://ki.se/en/bionut/welcome-to-the-lci-facility>
>
> Follow our microscopy blog!<http://microscopykarolinska.se/>
>
>
>
>
>
> När du skickar e-post till Karolinska Institutet (KI) innebär detta att KI kommer att behandla dina personuppgifter. Här finns information om hur KI behandlar personuppgifter<https://ki.se/medarbetare/integritetsskyddspolicy>.
>
>
> Sending email to Karolinska Institutet (KI) will result in KI processing your personal data. You can read more about KI’s processing of personal data here<https://ki.se/en/staff/data-protection-policy>.

[ICIT]

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Dear Sylvie.
For our MELC studies where hundreds of different primaries are applied on the same specimen and visualized sequentially we had to use FITC as it is bleachable. For labelling we used standard isothiocyanate chemistry and purified the antibodies with spin columns from Sigma. For studies verifying co-ip results with MELC approach we conjugated the co-ip bait as FRET acceptor (for FITC donor) with DyLight kit.

For assessment of functionality we used both biochemical and imaging approaches. Typically, however, we used more than one antibody against a protein and could evaluate the functionality as per co-occurrence. Only in very few cases we found out the antibody did not permit tagging. Unfortunately this was most of the time the case with commercial kits.

Best,

Mika