FAQ  •  Register  •  Login

MaxPar Antibody conjugation

Forum rules
It is fine to promote your company's reagents. Just make sure they are relevant to CyTOF, and do so in moderation and style :-)
<<

nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Wed Mar 08, 2017 12:58 pm

MaxPar Antibody conjugation

I have a couple of questions regarding MaxPar Ab conjugation.

1. How long would the antibody be stable in MaxPar CyTOF ready form (I have some purchased form Biolegend, which are described as partially reduced and not needing the buffer exchange steps in the Fluidigm labelling protocol) if stored at 4C? I have concerns that this may not be for long and one should try to conjugate as soon as possible.
2. When purifying the partially reduced antibody (after 37C incubation) is it add 300ul of C buffer to the antibody prep and then add this mixture to the 50kDa filter? Or is it add the antibody prep to the filter and then add the C buffer to the filter? The protocol isn't clear on this.
3. When washing the metal conjugated antibody there are repeat (total 4) washes with W buffer - by washes does the protocol mean to repeat the 10min centrifuge step and then wash?

Many thanks,
Naeem
<<

mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Mar 08, 2017 3:25 pm

Re: MaxPar Antibody conjugation

Hi Naeem,

1. I haven't seen the Biolegend documentation for their MAXPAR-Ready antibodies. However, from talking with them directly, the antibodies are being supplied in R buffer, rather than the more regular PBS. They are *not* partially-reduced; you still have to do the TCEP step.

2. Most people do the TCEP-reduction step of the antibody in the spin filter; if you're using regular antibodies in PBS, you've already had to do a couple buffer-exchange washes, so there's no need to transfer it back out of the spin filter for the TCEP step. Then, after 30min at 37C, you add the 300uL of C buffer to the 100uL of antibody+R buffer+TCEP and proceed to spin down/wash.

3. Yes. From the 300uL C buffer + 100uL antibody+R buffer+TCEP, you spin down. This should leave you with ~10uL of sample volume still contained in the spin filter (ie, "on top", rather than flow-through in the bottom tube). You then add 400uL of W buffer, spin down again for 10min to give you ~10uL volume. That's W-buffer Wash #1. Repeat for a total of 4 washes (ending in ~10uL).

It's this last ~10uL that you then add more W buffer (100uL, I believe) to and then take a protein concentration by NanoDrop.


Mike
<<

nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Fri Mar 31, 2017 2:10 pm

Re: MaxPar Antibody conjugation

Thanks again Mike.
I just did a conjugation this week and it worked though a few hiccups along the way. When retrieving the 3kDa filter the protocol doesnt explicitly say how to transfer the polymer from the filter. I assumed that you pipette 60ul of C-Buffer onto the 3kDa filter and then invert the filter and collect in a separate tube. Then add the mixture collected here to the 50kDa filter. Hope this is correct. Like I say it worked though yields were low at 35-40%.
I have another question on storage concentration. My antibodies are presently at about 0.32mg/ml and give good staining down to 0.3ul of antibody in a 100ul staining volume, which is approximately 1ug/ml. My antibody has some stabilizer (20% vol:vol) in already but would diluting the antibody further with more PBS stabilizer to a more convenient volume for pippeting also be helpful for greater stability?
Thanks,
Naeem
<<

mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Mar 31, 2017 3:12 pm

Re: MaxPar Antibody conjugation

Hi Naeem,

Generally, for convenience, I just pipet things out of the filter, rather than inverting and spinning. However, there's no reason you couldn't invert and spin to collect.

When I talked with Candor a couple years ago, they said that the more stabilizer there is, the better. I personally spec the antibody in W buffer, then dilute with an equal volume of stabilizer (eg, final 50:50 W:stabilizer). But last I heard, Fluidigm was doing 100% stabilizer....I don't think there's an issue with going to a higher stabilizer concentration than what you're left with.


Mike

Return to Reagents for CyTOF

Who is online

Users browsing this forum: No registered users and 13 guests

cron