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Bismuth conjugataion

PostPosted: Thu Apr 28, 2016 9:37 pm
by AdeebR
Hi all,
I'm sure most of you know that Fluidigm has started selling a few Bi209-conjugated antibodies, however they don't currently sell conjugation kits. Does anyone know if BiCl3 is compatible with the standard DN3/X8 polymers and conjugation kits?


Re: Bismuth conjugataion

PostPosted: Thu Apr 28, 2016 11:14 pm
by mleipold
Hi Adeeb,

At CYTO2013 in San Diego, Scott Tanner said that 209Bi is compatible with X8 and DN3. However, he said that there was some issue with the loading: apparently it's stable as a chelate when it's finally in, but the loading procedure had to be modified in some unspecified way. That's why Fluidigm doesn't sell a 209Bi kit.

I've heard of people being able to do it successfully, but I haven't heard of the details.

Sigma sells trace metals-basis Bismuth (III) chloride:
450723 anhydrous, powder, 99.999% trace metals basis
254142 99.99% trace metals basis

It's 100% 209Bi, so it would potentially be a very useful channel. If anyone has a loading protocol they'd be willing to share with the community, I think a lot of people would be interested.


Re: Bismuth conjugataion

PostPosted: Mon May 02, 2016 11:44 am
by ChristophS
Hi Adeeb,

I have sucesfully used Bi-Acetate (Sigma 401587-10G) but only after a couple of failures: it is insoluble in Ammonium Acetate and 2%HF, poorly soluble in 2% HNO3 but soluble in 1N HCl to 100mM. I then used 2ul of that with 100ul of solubilized polymer by first placing those 2ul in the 3k Amicon Filter and pipetting the polymer on as rapidly as possible followed by immediate vigorous suspension. Even this way you may still end up with some white precipitate (as soon as the HCl drops below 1N this will happen in a solution unless the chelator is there to pick it up) but that should come off in the 50k filter after the coupling. For the BiCl3 I would not know since I have not tried that.



Re: Bismuth conjugataion

PostPosted: Fri Sep 01, 2017 6:50 pm
by Chowduck

I gave 209Bi a try, happy with the initial result. Started by attempting to make a 50mM solution in L-buffer with bismuth (III) nitrate (Sigma 467839-50G). It seemed to dissolve then almost immediately formed a precipitate. I warmed the slurry to 55 degC for a few minutes which did not appreciably change anything, it's probably not necessary, then centrifuged and used the supernatant at 5x the recommended volume (adding 25 uL instead of 5 uL of metal solution to 100 uL of the solubilized X8 polymer) to conjugate anti-CD45. I hope to repeat this later this month and quantify the [Bi3+] present in the supernatant. Titration histogram attached. I knew there was Bi3+ in the L-Buffer supernatant since I could precipitate more of it out if I added concentrated HCL.

CD45-209Bi histo.png



PostPosted: Fri Jan 19, 2018 2:13 pm
by amaham

My intership is based on the use of mass cytometry. I tried to conjugate Bismuth like explained in the publication of Han et al.Cytometry part A (2017) but it didn't worked. :cry:
I wonder if this is due to the acid used to pre-load Bi on the polymer ?
Do someone here have tried to do it ?
Do you have any suggestion ?

Thanks :)

Re: Bismuth

PostPosted: Fri Jan 19, 2018 7:19 pm
by mleipold
Hi Mahamad,

Could you give us more information about what you mean by "it didn't worked"?

For example, did you not get any Bi signal? Did you not get any protein recovery? and so forth.


Re: Bismuth

PostPosted: Fri Jan 19, 2018 7:30 pm
by Ofir
Moved thread to relevant topic.
Please continue the conversation here.

Re: Bismuth conjugataion

PostPosted: Fri Jan 19, 2018 10:50 pm
by GregBehbehani
We have followed the Han protocol and it worked for us. We also just threw Bi (BiCl and BiAcetate) into the regular labeling protocol and it works, but the Bi precipitates and the labeling index isn't very good. I do believe the extra washes (after loading the polymer) in the Han protcol are important, however, as the acid would be expected to prevent the coupling reaction if you don't wash it away. This might be where you had an issue.

As per Mike's comment, it's hard to tell you much more without some more information.



Re: Bismuth conjugataion

PostPosted: Mon Jan 22, 2018 8:30 am
by amaham

By "it didn't work" I meant that I had no signal on CyTOF ; however I got protein recovery.
I modified some MAX PAR conjugation protocol steps to stick with the publication protocol :
- like using 5% HNO3 to pre-load instead of L-Buffer ;
- change the volume or the buffer using for purify Bi-loaded polymer.

Re: Bismuth conjugataion

PostPosted: Mon Jan 22, 2018 8:37 am
by amaham

And thanks you all fo your reply !

- Ahlam