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Glycine in purified antibodies for conjugation

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ghowell

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Joined: Wed Nov 26, 2014 3:18 pm

Post Mon Oct 21, 2019 12:10 pm

Glycine in purified antibodies for conjugation

Dear all,
I'm pretty sure it makes no difference but would the presence of 0.1M Tris-Glycine in a purified antibody prep have any affect on the efficiency of conjugations?
Thanks
Gareth
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AndrewD

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Posts: 9

Joined: Tue Feb 20, 2018 7:18 pm

Post Mon Oct 21, 2019 1:34 pm

Re: Glycine in purified antibodies for conjugation

If you are conjugating via thiol groups (X8 and MCP9 polymers) then tris and glycine shouldnt affect this - you cant have any glycerol or carrier protein present though. The protocol recommends swapping the buffers to those supplied in the conjugation kit anyway using 50KDa filters so you would normally change buffers before conjugation anyway.
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DMcDonald

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Posts: 11

Joined: Thu Nov 26, 2015 10:14 am

Post Mon Oct 21, 2019 2:21 pm

Re: Glycine in purified antibodies for conjugation

Hi Gareth,

Yes we've done loads in tris-glycine. Andrew: we've also done a lot in glycerol too with the X8 polymer and haven't had any issues with these. It's actually quite amazing what you can get away with...I recently did a "ready to go" one that was 7 mL in volume and bright green too...it took a while but worked!

Best wishes,
David
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mleipold

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Posts: 1751

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Oct 21, 2019 3:16 pm

Re: Glycine in purified antibodies for conjugation

Andrew:

You said that you don't recommend having glycerol around: is there a particular reason? While glycerol is viscous, it can be removed during the R buffer wash step, and doesn't have any thiols (unlike most carrier proteins), so it shouldn't chemically interfere with the reduction and conjugation.

I haven't had any issues with glycerol.


Mike
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AndrewD

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Posts: 9

Joined: Tue Feb 20, 2018 7:18 pm

Post Wed Oct 23, 2019 10:05 am

Re: Glycine in purified antibodies for conjugation

Hi,

I wouldnt have thought glycerol would have a big affect on the conjugation either but fluidigm seem to recommend its absence according to their protocols: "Antibodies used with this kit must be purified, glycerol-free, and carrier-free (no BSA,
hydrolyzed protein, or gelatin for stabilization)." Glad to know that other people havent had problems with it being present.

BW
Andrew
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mleipold

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Posts: 1751

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Oct 23, 2019 1:32 pm

Re: Glycine in purified antibodies for conjugation

Hi Andrew,

FYI, gelatin generally won't interfere with the conjugation chemistry, as all forms of it I've looked up lack Cys.

However, it *will* usually mess up your Nanodrop protein calc. And, some people have found washing and recovery issues if the residual volume gets too viscous (ie, if your filter pores are small enough to retain gelatin).

So, it's undesirable, but not a complete no-go like BSA.

Mike
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Verhoeff

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Posts: 11

Joined: Wed Mar 02, 2016 10:25 am

Post Thu Oct 24, 2019 6:34 am

Re: Glycine in purified antibodies for conjugation

Hi all,

While gelatin does not contain cysteines, there are limits to how much gelatin the filters can handle. A BD anti-CD34 clone that we tried came in 50% gelatin. Residual volume in a 50kd filter would not go lower than 200uL, even after multiple washes with R buffer.

Cheers,
Jan

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