What is the purpose of the Q spin filters used in your HRP kit?

These are ion exchange columns with a positive charge on the resin surface. The pH of one of the solutions in the kit is used to alter the charge on the conjugated antibody – above the pI of the antibody, the antibody-HRP complex will have more negative charges, and will bind to the resin. At this pH, the HRP is still positively charged (a higher pI) and will pass through the column, thereby separating the free HRP from the Ab-HRP conjugate. Then, the pH is changed again, below the pI of the antibody, thereby increasing the positive charge on the Ab-HRP conjugate, such that it passes through the column. The same strategy is used in the PE kit.

Tell me more about the Zeba columns (from Pierce website).

Zeba Spin Desalting Columns contain a proprietary high-performance desalting resin that offers exceptional desalting and protein-recovery characteristics compared to other commercially available resins. Even very dilute (25 µg/mL) protein samples can be successfully processed to obtain greater than 95% retention (removal) of salts and other small molecules (< 1,000 MW) and good recovery of proteins and other macromolecules (>7,000 MW). Despite claims, other commercially available resins perform only satisfactorily in spin columns with sample of high concentration (>250 µg/mL) over a narrow window of sample volumes. The proprietary Zeba Micro Spin Desalting columns has exceptional protein recovery and desalting characteristics with >95% retention of salts and other small molecules of < 1,000 MW and are recommended for processing compounds >7,000 MW.”

What is the purpose of the desalting columns used in many of your kits (Zeba spin columns)?

These columns are size exclusion columns with a MWCO of 7 kDa. These are used to exchange buffers – to put the biomolecule to label in the correct buffer, such as a modification buffer. Small contaminants (e.g., various buffer components under 7 kDa will go into the bead matrix or resin, and retained, as larger molecules flow through the column. This both removes buffer components which might interfere with the reaction and puts the biomolecule of interest into a new buffer.

We sometimes notice a precipitate in your 10X buffers. Are the buffers OK to use??

The 10X conjugation buffer and 10X modification buffer contain a high concentration of salt, which can precipitate out of solution and form crystals at room temperature, and especially when stored at 4 degrees. This is normal, and occurs frequently. There is nothing wrong with the buffers. You can heat the 10X solution to 40 degrees and vortex, to get all of the salt crystals back into solution. Then, you can use the buffer to make your 1X dilution.

How do I dissociate my antigen from my antibody?

Because the binding is so tight, harsh methods are typically used to dissociate an antibody from its target antigen. One can use 0.1 M glycine, 0.5-1% Triton X-100 at pH 2.8 or boil in protein lysis buffer at 95-100ºC 5–10 min. Some vendors market a less harsh method of separating the two biomolecules (Pierce sells a gentle Ag/Ab elution buffer).