FAQ  •  Register  •  Login

Labeling an antibody preconjugated to PE or FITC

<<

Verhoeff

Participant

Posts: 16

Joined: Wed Mar 02, 2016 10:25 am

Post Mon Jun 06, 2016 1:33 pm

Labeling an antibody preconjugated to PE or FITC

Hi all,

At our group we're labeling the antibodies ourselves. Nearly all antibodies are available in purified form, but for one it is only available already conjugated to either PE or FITC. Does anyone have any experience with conjugating a metal isotope to an antibody+fluophore? We reckon once the labeled cell hits the plasma torch it shouldn't matter for your measurement but we're not sure about the conjugation process.

Any feedback is much appreciated!

Greetings,
Jan
<<

Ofir

Master

Posts: 75

Joined: Thu Nov 07, 2013 12:46 pm

Location: US, CA

Post Mon Jun 06, 2016 9:47 pm

Re: Labeling an antibody preconjugated to PE or FITC

Jan, Fluidigm sells secondary antibodies anti-FITC, anti-APC, and anti-Biotin.
Would a secondary antibody approach work for you?
<<

mleipold

Guru

Posts: 2156

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Jun 06, 2016 9:56 pm

Re: Labeling an antibody preconjugated to PE or FITC

Hi Jan,

I haven't tried MAXPAR labeling an antibody already labeled with a fluorophore, so I can't give any tips.

The issue would be whether the fluorophore conjugation would use the same chemistry as the MAXPAR. For example, ThermoFisher sells this product to label a protein with Alexa488, using maleimide: https://www.thermofisher.com/order/cata ... uct/A10254 That would conflict with the MAXPAR chemistry, as your thiols would be already occupied.

Taking a quick look at Google for "FITC conjugation antibody", it seems like most of the kits use amine chemistry, rather than the thiol chemistry used in MAXPAR. If that's what your commercial-antibody manufacturer used, then it's worth a try.


Ofir's point about using the antibo-fluorophore antibodies as a secondary does work well....I've used aAPC and aPE, and I know people who have had good success with aFITC.


Mike
<<

sfauteux

Participant

Posts: 17

Joined: Tue Feb 23, 2016 2:18 pm

Post Tue Jun 07, 2016 6:13 am

Re: Labeling an antibody preconjugated to PE or FITC

Hi Jan,

I never have done this but i'm very interested in the result if you try it. Perhaps you will need to exchange the buffer of the Ab.

Looking forward to ear from you

Best of lucks.

Return to Introduction to mass cytometry

Who is online

Users browsing this forum: No registered users and 3 guests