Griffin:Antibody Protein Coupling (F:P) Ratios
Antibodies from the same manufacturer can exhibit batch to-batch variation. In addition, some control antibodies are not marketed at the same concentration as the test antibody. Many available reagents have high F:P ratios and are better avoided if alternatives exist (over- conjugation can produce highly charged acidic species which tend to stick non-specifically to cells, especially if they are fixed). It is important to establish the correct working dilutions for every new antibody, including those from different batches, for each detection system.
When tagging antibodies what is important is optimizing the tag:antibody ratio so that the maximum number of tags are coupled to the antibody without affecting its antigen binding activity. This is more important than the potential number of tags that can bind to every SH or NH2 groups available. For standard tags like FITC, Biotin, HRP, etc. there are standard coupling procedures to achieve this. For others, an optimization process may be required.
Most antibody conjugates are done by using the NH2 groups from amino acid side chains in the IgG proteins (i.e., primary amines from lysine). This seems to be less disruptive for the IgG. There are some chemistries that take advantage of SH groups in IgGs. The SH groups are not readily available and need to be produced by reducing disulfide bonds (S-S) with mild reducing agents prior to coupling. This has the potential of disrupting the IgG secondary and tertiary structures and potentially harming its antigen binding activity. There are 16 Disulfide bonds in IgGs, with a potential to form 32 SH groups. Average number of lysine residues in IgG (there are a lot of them)
Suitable coupling ratios
- 3 to 8 biotin per IgG molecule (biotin MW: 244); (long arm) N hydroxysuccinimide ester
- 4 to 7 FITC per IgG molecule (FITC MW: 390)
- 1 to 2 HRP per IgG molecule (HRP MW: 40,000)
- 1 APC per IgG molecule (APC MW: 100,000)
- ~1 PE per IgG molecule (PE MW: 240,000)
- AF405: 1-3 moles of dye: mole of IgG
- AF488: 4-9 moles of dye: mole of IgG
- AF647: 3-7 moles of dye: mole of IgG
Peptide to Solid Phase Coating
- Coating peptides can be prepared as follows:
- Free peptides, 2.5 ug/50 ul solutions (or suspensions for some peptides of low solubility) in either PBS or 0.05 M sodium carbonate buffer, pH 9.6
(SCB). Peptide-BSA conjugates dissolved in 0.05 M NH4HCO3, pH 7.8 (0.3 mg per ml), centrifuged and the supernatants used for plating.
- The following order of steps, each of which succeeded by washing of the wells with PBS (5x each).
- PVC 96-well microtiter plates were coated for 3hr/ 37C with 50 ul/well of antigen solution.
- After coating, wells were blocked for 1 h at 37C with 100 ul of 0.5% casein in PBS to reduce non-specific binding to the wells.
- After washing with PBS, 50 ul of anti-peptide antiserum, prediluted 1:500, can be added to each well / incubated for 2 h at 37C.