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Metals tubes

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mmeyrand

Participant

Posts: 6

Joined: Tue Nov 26, 2013 8:20 am

Location: Marseille, FRANCE

Post Tue Nov 26, 2013 10:44 am

Metals tubes

Hi,
We got 9 months ago 20 tubes of metals containing each 20µl (good for 4 conjugation of 100µg).
We are not using the metal tube in one time, but in 4 different times, for 4 different antibodies.
For some of them, I recently realized I will not have enough volume to perform the fourth conjugation. Maybe there is a small evaporation, meaning that the metal get concentrated over the time (this could maybe explain a protein precipitation during conjugation? excess of metal is washed on the 3kDa amicon before combination with the antibody, but who knows?)

Yes I keep them at 4 degree, Yes I keep them cool when taking them out of the fridge, yes I centrifuge them before opening the tube.

I am wondering if you guys get the same problem sometimes and if it could be a good strategy to aliquot the metal (5µl in smaller tube) immediately after reception.
Thank you for your help
Cytofely
Mickael
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Nov 27, 2013 7:07 pm

Re: Metals tubes

HI Mickael,

We have seen some evidence for minor evaporative volume loss over long storage, even at 4C.

I personally would not recommend aliquotting them into smaller volumes. The smaller the volume, the faster the evaporation.

However, if you make the single-use aliquots as you propose, you could always add 5uL of MilliQ water at the time of use to make sure that you have all the metal salt dissolved. The exact *volume* of salt solution added to the chelation step doesn't matter, just the number of moles of metal ion. Therefore, whether you have a completely dry tube and you wind up with 5uL of 50mM stock like normal, or whether you have a partly dry tube (2.5uL) that then gives you 7.5uL of 37mM stock, the fact that this is the same number of *moles* is all that is important.


Mike
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mmeyrand

Participant

Posts: 6

Joined: Tue Nov 26, 2013 8:20 am

Location: Marseille, FRANCE

Post Fri Dec 06, 2013 8:49 am

Re: Metals tubes

thank you Mike for all your answers.
Regarding my other question concerning protein precipitation, do you think both problem could be related? for me, even if I doubled the quantity of metal because of evaporation, metal concentration is not high enough to precipitate my protein during conjugation,right?
Thanks
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Dec 06, 2013 4:09 pm

Re: Metals tubes

I think it's *unlikely* that doubling the metal ion concentration would be enough to precipitate the polymer. But I have not tested this directly.

Remember, you wash away excess free metal *before* adding the metal-loaded polymer to the partially-reduced antibody solution. Therefore, the antibody shouldn't precipitate, as it should never "see" excess free metal.
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richardellis

Participant

Posts: 15

Joined: Mon Dec 09, 2013 11:03 am

Post Mon Dec 09, 2013 11:59 am

Re: Metals tubes

Hi Mickael, we have seen the same - in our 4-reaction tubes supplied last year, even when unopened, there is/was typically only 17 or 18 ul available volume.

Tubes supplied more recently have had a small excess (as is typical for many other reagents you buy) so perhaps this was changed to allow for losses on tube walls, evaporation etc. If so I don't think there will be any problem going forward. As Mike said, I wouldn't advise smaller aliquots.

There are a couple of cases where I did have to use slightly less metal volume for the conjugation (whether it is more concentrated or not I don't know*). That is, I used perhaps 3-4 ul rather than 5 until the next order for metals arrived (also rinsing the tube with the polymer solution to maximise recovery on the last metal-labeling. Not ideal, I know, but in those cases the actual staining was okay.

*Of course I never checked then or now the metal solution concentration as supplied - the antibodies were required urgently so I made the assumptions that the kit protocol is tolerant of fluctuation in concentration to some degree, and the metal is supplied in excess. As with you, we only noticed the shortage when our first batches of metals were used 3 or 4 times.
Guy's Hospital flow core, London

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