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Conjugation for cytof

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Kjwaller

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Posts: 11

Joined: Sat Aug 27, 2016 6:56 am

Post Mon Sep 05, 2016 11:03 am

Conjugation for cytof

Hi, I was hoping someone may be able to help me. I recently conjugated my antibody, using a "maxpar ready" one (1mg/mL in 100uL). My recovery was ok, not fantastic but as my first attempt not bad. My final concentration is 0.6mg/mL in W buffer, it says to store it at 0.5mg/mL in antibody stabilisation buffer but that would only be the addition of an extra 20uL in stabilisation buffer. I know I can dilute it more and I have read elsewhere that it may be stable for a couple of months or more in W buffer but ideally I would rather give it the best shot and be in the buffer recommended at 0.5mg/mL. My problem is I have no idea whether spinning it down is suitable and if so at what speed etc. I surely can't be the only person that has come across this?

Thanks,
Kate
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mleipold

Guru

Posts: 1778

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Sep 07, 2016 4:32 pm

Re: Conjugation for cytof

Hi Kate,

At the HIMC, we usually dilute the recovered antibody with stabilizer to 50/50 W/stabilizer (ie, equal volume of stabilizer). We have found that that's sufficient for stability to at least several months.

Candor says that the more stabilizer you use, the more stable your stock. Last I heard, Fluidigm antibodies are supplied in 100% stabilizer: they resuspend in W buffer, take a Nanodrop reading; then spin down again and resuspend in stabilizer to the original volume. I personally find that to be a hassle, and the 50% route works well. I also personally don't bother with making all of my antibodies the same concentration: I don't use them all at the same titer, and instead I use an Excel sheet to calculate the volume of each stock I need for my cocktail.


If you *are* going to spin down, then use the same speed as you did during the conjugations.


Mike
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RSchackmann

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Posts: 9

Joined: Fri May 27, 2016 5:26 pm

Post Fri Sep 09, 2016 6:14 pm

Re: Conjugation for cytof

Hi Kate,

I also dilute my ab's 50/50 with candor antibody stabilizer (pbs) for long term storage. Like Mike, I also don't bother to set the concentration to a certain value and I also use an xls sheet to calculate how much of each antibody is required for a staining.

,
Ron
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Kjwaller

Participant

Posts: 11

Joined: Sat Aug 27, 2016 6:56 am

Post Wed Sep 28, 2016 2:22 pm

Re: Conjugation for cytof

Thank you Ron and Mike, I will stick with your methods then.

Kate

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