proteintech antibodies

[DanaiBD]

Hello everyone,
I wanted to ask whether any one has experience with proteintech antibodies. I am interested in conjugating one, but it contains (https://www.ptglab.com/products/DIO2-An ... 3-1-Ig.htm) 50% glycerol (pH 7.3) and I am unsure whether that could cause an issue.
Thank you in advance for your help

[mleipold]

Glycerol will not interfere with the chemistry of the conjugation.

However, some people have reported that the viscosity of high-percentage glycerol solutions makes it harder to mix or recover from the spin filter. In most cases, doing additional buffer washes to wash out the glycerol is good enough to address this issue.


Mike

[marthabrainard1]

Hi Dana,

I just conjugated an antibody that was stored in 50% glycerol last week. Prior to the conjugation, I mixed the antibody 1-1 with PBS and spun it down (12,000g 10 minutes) in a 30 kDa filter, it worked nicely (as Mike already mentioned!)

[DanaiBD]

Thank you very much everyone

[mds4z]

Dana

We are planning to try conjugating several Proteintech mabs for IMC. Many are in glycerol as you mentioned, much higher price to purchase in PBS only. You mentioned 1-1 PBS dilution and spin below. Is anyone doing any other steps in the conjugation steps differently when the glycerol is present? Protocol to share?

Another question, what is the largest amount of mab we can label with the maxpar kits. I believe the kits recommend 100-200ug of antibody for labeling, but wondering if people are labeling with more.

Thanks
Mike

[CRStevens]

Hey Mike,

We routinely conjugate 400ug at a time. Sometimes for large pools I'll do side-by-side 400ug reactions for a total of 800ug and pool at the end.
Really it is the same as the current protocol, except you add the 20uL of metal to the 95uL of L-buffer and the amount of C-buffer you resuspend you metal-chelated polymer for the conjugation is scaled.
My personal opinion is I get much higher yeild from doing bulk conjugations like this and I also still see great metal numbers when I quantify my antibodies (not required to do).

This is the protocol we use that was shared with us from a former colleague at DVS:

Each MaxPar kit standard size is sufficient for up to 4 rxn of 100 ug each (so total of 400ug). Let’s assume you would want to feasibly go as high as 400ug each prep (one kit). To do this, we suggest the following based on our experiences and generally following the standard 100 ug reaction protocol for incubation and centrifugation times:

A. Preloading Polymer with Lanthanide:
a. Resuspend the 4 vials of polymer into total of 95 uL L-Buffer (use 95 uL to resuspend one tube then transfer to the next to resuspend, etc) such that the contents of the 4 vials of
polymer ends up in one tube in 95uL of L-Buffer.
b. Adjust the amount of metal to match the scale of the antibody (so 400 ug of antibody would need 20 uL of the metal).

B. Buffer Exchange & Reduction of Antibody:
a. No change to this part of the protocol for up to 400 ug. It can be done in the same 50KDa filter and keep the volumes the same as if the quantity of Antibody were only 100 ug.

C. Purify metal-loaded polymer and reduced antibody:
a. No change to this part of the protocol for up to 400 ug. The same 3 KDa filter can be used and the volumes and centrifugation steps stay the same as if the quantity of Antibody
were only 100 ug.

D. Conjugation of Antibody with Metal Loaded Polymer:
a. Resuspend the metal-loaded polymer (4X) in 200 uL of C-Buffer.
b. No change in the centrifugation and incubation steps. Keep those the same as if the quantity of Antibody were only 100 ug.

E. Wash Metal Conjugated Antibody:
a. No change to this part of the protocol (keep as it the quantity of antibody were only 100 ug).
F. Recover Metal Conjugated Antibody:
a. Resuspend the recovered antibody in 300 uL of W-Buffer (bring volume up with antibody preservation buffer to 500ug/mL)
b. Measure by Nano-drop as usual.