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Maxpar W-buffer and stabilization buffer

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vvanunen

Participant

Posts: 9

Joined: Tue Nov 26, 2013 12:49 pm

Post Tue Feb 18, 2014 3:00 pm

Maxpar W-buffer and stabilization buffer

Hi all,

I have a question regarding long term storage of stock antibody conjugated with MaxPar kit.
The final steps your antibody is collected in 100 uL W-buffer according to DVS protocol. Do you get rid of the W-buffer, to dilute down in stabilization buffer? Do you mix stabilization buffer with W-buffer containing your antibody?

What are your experiences?

Thanks,
Vincent
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elinastar

Participant

Posts: 19

Joined: Sun Nov 17, 2013 1:46 pm

Post Thu Feb 20, 2014 7:22 am

Re: Maxpar W-buffer and stabilization buffer

Hi,
We collect in W-buffer, measure protein concentration using W-buffer as a blank, and then dilute two-fold in stabilizer. We take this dilution into account when we calculate antibody concentration for titrations. After we decide on optimal staining concentration we further dilute in antibody stabilizer.
Hope this helps.
Elina
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Feb 20, 2014 4:16 pm

Re: Maxpar W-buffer and stabilization buffer

Hi Vincent,

At the Stanford HIMC, we generally elute the protein in 100uL W buffer, spec it for protein concentration, then dilute it with an equal volume of antibody stabilizer (final 200uL total, at 50% of original concentration; 50/50 W buffer/stabilizer). Most of the time, we don't readjust the concentration after that, we just add more or less volume to the antibody cocktail if we have to re-titer.

At 50/50, we get reproducibly at least 4 months out of all of our antibody stocks. There are a few that start to decrease around 4-5 months, but we run enough experiments that we usually use the stock up before then. Many antibodies are stable for 6+ months.

According to the stabilizer buffer manufacturer, the more stabilizer, the better. They do recommend re-spinning after protein determination, and resuspending in 100% stabilizer. I know from talking with DVS, that this is what *they* do...all of their preconjugated antibodies are in 100% stabilizer. DVS has also done lifetime stabilities (accelerated, and regular), and they guarantee 6 months. However, I've heard that they're good for longer than that: DVS just doesn't want to officially guarantee that.


Mike
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Ofir

Master

Posts: 75

Joined: Thu Nov 07, 2013 12:46 pm

Location: US, CA

Post Thu Feb 20, 2014 6:34 pm

Re: Maxpar W-buffer and stabilization buffer

Hey Mike,
Could you specify which antibody clones had poor stability in your hands?
This is important because some antibodies are used less frequently than others at the Technion.

Thanks!
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Feb 20, 2014 11:16 pm

Re: Maxpar W-buffer and stabilization buffer

Hi Ofir,

Out of my panel of 23 markers (at the time), there were only a couple that had a lifetime of 2months only, before the stabilizer buffer.

The main one that immediately springs to mind is R&D Systems' CD85j (ILT2/LILRB1) clone #292319. That's the best CD85j clone we've found yet for resolving the CD85j-dim CD8+ cells.

However, since it's now 50/50 W/stabilizer, it gets at least 4 months. Since I use it at a higher titer, I'm almost always remaking it before it hits the 4mo limit.


Mike
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Ofir

Master

Posts: 75

Joined: Thu Nov 07, 2013 12:46 pm

Location: US, CA

Post Mon Feb 24, 2014 7:26 pm

Re: Maxpar W-buffer and stabilization buffer

Thanks!

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