Conjugate storage concentration?

[vglui510]

Hello,

I prepared some antibody conjugates, measured protein concentration via Nanodrop, and adjusted the final concentrations to 0.2 mg/mL with 50/50 W buffer and stabilization buffer. When I titrate some of these conjugates, I find that the ideal titer is super low, in the range of 0.1 uL/100uL rxn volume. To make this manageable, I want to dilute the stock of conjugate ~ 5-fold with 50/50 W buffer and stabilization buffer.

Is it ok to dilute the 0.2 mg/mL stock of conjugate that much?

Would there be an issue of protein sticking to the side of the eppi tube?

Thanks

[MCOlivier]

Hi,

I don't think it's a good idea to dilute your AB that much. This increases water activity and accelerates the "natural" dissociation of any compound dissolved in water.
I would either pipet carefully with accurate pipets, or make a temporary 10x dilution aliquote for each experiment prior pipeting ^^.

regards

Olivier

[mleipold]

Hi Victor,

I personally wouldn't worry as much about diluting things to a workable concentration. The stabilizer buffer contains unspecified protein in it, which helps prevent at least most of the dilution-related activity loss (whether it's related to molecular crowding or not), or sticking to the tube.

I remember when we first started using it, I talked with Candor and they suggested using it as concentrated as possible (ie, using 100% stabilizer rather than 50% stabilizer/50% W buffer) for maximum stability. So, if anything, I'd recommend that you dilute your ultra-active stocks with 100% stabilizer rather than 50/50.

But yes, another way to do it is make an initial day-of dilution as Olivier suggested, and then dilute that appropriately into your cocktail.


Mike

[Jahangir]

Hi all,

I'm not too sure if you do this already, but you can also use protein specific, lo-bind eppendorfs to ensure that you get all the antibody from the eppendorf. I buy these -> https://www.fishersci.co.uk/shop/produc ... e/10708704.

Hope that helps!

Jahangir