FAQ  •  Register  •  Login

Maxpar conjugation kit

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 :-)
<<

mmeyrand

Participant

Posts: 6

Joined: Tue Nov 26, 2013 8:20 am

Location: Marseille, FRANCE

Post Tue Nov 26, 2013 10:25 am

Maxpar conjugation kit

Hi everybody,
I am wondering if it happened to you to get a kind of precipitate in your final elution when doing a conjugation using Maxpar procedure? Nanodrop indicates there is no protein in my elution !

We perfectly follow the procedure, I checked every paramaters such as temperature, pH, concentration of the initial antibody, incubation time, etc... everything look OK
These purified antibodies have been already conjugated in different metals. The metals also have been used with success for others conjugations.
Polymer lot is still the same, and since we use 4 or 8 polymer tubes at the same time, we never have to store them outside the sealed bag.
We work at room temperature, and I decrease a little bit the time/speed with the centricon columns to make sure not to dry my protein (even if there is a safe volume at the bottom of the column).

I don't know at which step my prtoein/salts precipitate, I visualize it only at the final elution when I turn over the column.

Thank you very much for your help
Cytofely
Mickael
<<

mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Nov 27, 2013 7:02 pm

Re: Maxpar conjugation kit

HI Mickael,

There are some antibodies that are more difficult to conjugate. For example, the CCR7 clone I use (clone 150503, from R&D Systems) is one that often has lower recovery, and occasionally just "fails" (no protein content, as measured by Nanodrop).

However, on occasion, you just get a bad lot of antibodies. I've had bad lots of CD20 antibody (Biolegend) and CD56 antibody (BD). Conjugations had worked fine multiple times before, but then failed repeatedly. However, as soon as I switched to a new lot, they worked again.


Much less likely: if you have not washed away all the free metal from the polymer loading step, residual metal ions *can* occasionally cause proteins to precipitate. But if you're using DVS-prepared metal stocks, this is highly unlikely.

Similarly, lanthanide phosphates are insoluble: if you haven't washed away all of the PBS from your commercial stock, you can get precipitates.

Finally, occasionally, too much TCEP can cause protein (antibody) denaturation. Again, unlikely if you're following the DVS labeling protocol.


Mike

Return to Reagents for CyTOF

Who is online

Users browsing this forum: No registered users and 11 guests