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CyTOF labeling using higher amount of Ab

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sjp2016

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Joined: Thu Sep 22, 2016 8:31 pm

Post Thu Sep 22, 2016 9:42 pm

CyTOF labeling using higher amount of Ab

Hello,

Has anyone ever tried to label more than 100 ug of Ab per reaction using Maxpar Ab kit?

We have labeled up to 300 ug of Ab per reaction. At first we did not increase the volume of any buffers proportionally and got reasonable recovery (~ 70%). Recently when labeling 300 ug of Ab we decided to increase the volume of 4 mM TCEP buffer and the C-buffer used to resuspend the lanthanide-loaded polymer to 300 ul and 180 ul, respectively (i.e. three times the volume used for 100 ug of Ab) and saw the recovery rate plunge.

Does anyone know why that is the case?

Thank you!

S.J.
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BjornZ

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Posts: 33

Joined: Fri Jul 10, 2015 1:04 am

Post Mon Sep 26, 2016 4:56 am

Re: CyTOF labeling using higher amount of Ab

Hi S.J.,

We've routinely labeled up to 1 mg of antibody, and basically the only change we had to do was increase the polymer and metal proportionately. All other volumes can stay the same, including the volume in which you resuspend the polymer. You may benefit from doing an extra W buffer wash or two at the end to help remove excess polymer. Usually we get ~80% recovery or better for these large conjugations.

There's a protocol on the Nolan lab website that covers 100 to 1000 ug reactions: https://nolanlab.stanford.edu/protocols last one under "CyTOF Protocols."

(As far as why your recovery plunged, I'd assume one of those changes caused the Ab to precipitate, but don't know for sure.)

Zach
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Chowduck

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Joined: Wed Nov 19, 2014 4:39 pm

Location: Toronto, Canada

Post Mon Sep 26, 2016 3:29 pm

Re: CyTOF labeling using higher amount of Ab

Hi S.J.,

I would back off the 300ug a little, your staining might be "dimmer". I use 150ug as a balance of risk/reward that I’m comfortable with. Here are some calculations you may find helpful:

Polymer: 11kDa, 100ug/rxn, (~10 chelation sites/polymer) = 9.09x10^-9 mol polymer/rxn

Antibody: 150kDa, 100ug/rxn, (4-6 conjugated polymers/antibody) = 6.67x10^-10 mol Antibody/rxn

Metal: 50mM, 5uL per rxn = 2.50x10^-7 mol metal/rxn

Per reaction this means that there are 13.64 available polymers per antibody, at the high end if we expect 6 polymers to bind then this would saturate at 227ug of input antibody assuming the reaction exhausts the entire supply of polymer. (If 4 polymers then 340ug of antibody, but why aim low. Also you might be left with free -SH groups). At 5uL the metal is in a 2.8 fold excess to chelating sites on the polymer so don’t worry about that.

Another observation I’ve made is that most of the antibody loss is going to happen in the first few spins meaning that if you’re loading 300ug, by the time that antibody comes into contact with the polymer you might be looking at ~250ug remaining. If the recovery is poor and I have starting material remaining I will run ~5-10ug on an SDS-PAGE to get a sense of purity or presence of peptide fragments that may wash through the filter. I’m not certain why the increased volume affected your recovery unless there was a filter specific defect. If you repeat it, pull some antibody out after the reduction, prior to the conjugate to look at on a SDS-PAGE maybe it was over-reduced?

Regards,
-Greg
UHN/Sickkids FCF
Toronto, Canada
The SickKids - UHN FMCF
Toronto, Canada
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sjp2016

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Joined: Thu Sep 22, 2016 8:31 pm

Post Tue Sep 27, 2016 3:05 pm

Re: CyTOF labeling using higher amount of Ab

Hi Zach,

Thank you for the link to the protocol.

Your protocol is different from the Kit protocol. I want to make sure I understand it correctly, can you please clarify some points for me:
(1) Step 2: when you scale up, do you resuspend the polymer in a final volume of 95 ul (regardless of the number of MaxPar tubes used per Ab) or 95 ul for each MaxPar tube used? If it is the former, do you just add the corresponding volume of metal to the final PCR tube?
(2) Step 4 (loading Polymer with metal): Why do you incubate the tube at room temp instead of 37 degree C?
(3) Step 19: Why do you resuspend the metal-loaded polymer in 200 ul of C-buffer instead of 60 ul?

Thank you so much for your help.

s.j.
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BjornZ

Contributor

Posts: 33

Joined: Fri Jul 10, 2015 1:04 am

Post Tue Sep 27, 2016 4:16 pm

Re: CyTOF labeling using higher amount of Ab

Hi S.J.,

(1) I add 95 ul to one polymer tube, then transfer the entire 95 ul to the next tube, and repeat for all remaining polymer tubes; the final total volume is 95 ul. The metal volume is per the table (i.e. 5 ul of 0.05 M solution per 100 ug of antibody).

(2) 37 degrees is fine as well. If I recall, 37 degrees is specified to provide a more consistent temperature day-to-day than "room temp."

(3) Unfortunately I forget the origin of this difference. It might be important to prevent precipitation of the antibody when combining with the metal+polymer.

This protocol has served us well through more than 250 mg of conjugations. If you try it, perhaps you can post your experience here as well.

All the best,
Zach

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