It is critical to purify proteins (e.g. antibodies) before conjugation to nanoparticles. Commercial antibodies may contain salts and protein additives for stabilization (e.g. BSA), amines as a preservative (e.g. sodium azide), or amines in the buffer (e.g. Tris) that need to be removed before conjugation.
The filters provided by nanoComposix (Millipore-Sigma Cat # UFC5010) have a 10 kDa molecular weight cut-off for antibody purification and are suitable for purifying or concentrating proteins larger than 10 kDa, while filtering out small molecules and free amines such as sodium azide or transferring to a suitable buffer. These filters will not remove unwanted stabilizing proteins >10 kDa, such as BSA. Use appropriate affinity columns to isolate the antibody from stabilizing proteins if needed. If Tris or another amine containing buffer is used to elute the antibody from the affinity column during isolation from stabilizing proteins, the antibody will need to be purified a second time to transfer it to a suitable amine-free buffer.
It is important to note that the antibody purification referred to in this procedure is different than affinity purification during antibody development and processing. Although the antibody may have been affinity purified, it needs to be purified and transferred into a suitable buffer free of additional amines. Even a small concentration of sodium azide (NaN3) will interfere with the efficacy of conjugation.
Note: The filter can hold up to 500 µL. If the volume to purify exceeds this capacity, you can centrifuge to concentrate and add additional unpurified antibody before continuing with the wash steps. If the starting antibody volume is minimal, make up the volume by adding buffer up to ~450 µL total volume.
Note: There will be a small amount of solution that does not go through the membrane. This contains the antibody. Spinning until all the solution has gone through may cause adsorptive loss of antibody on the membrane.
Note: For optimal recovery, perform the reverse spin immediately. The cap may be cut off with scissors before spinning.
Use A280 method, BCA, or Bradford assay to confirm the concentration of starting material and final purified material to determine yield.
Low sample recovery in concentrate may be due to adsorptive losses, over-concentration, or passage of sample through the membrane. To maximize sample recovery:
If the sample appears to be passing through the membrane, choose a lower nominal molecular weight limit (NMWL) Amicon Ultra-0.5 filter unit.
For technical support, please contact us.
Published June 2017
Version 1.2