Griffin:Antibody Protein Coupling (F:P) Ratios: Difference between revisions
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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. Ideally the F:P ratio (fluorochrome : protein ratio) should be 1:1. 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. Below are some examples of suitable coupling ratios for various detection molecules to the antibody, | 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. Ideally the F:P ratio (fluorochrome : protein ratio) should be 1:1. 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. Below are some examples of suitable coupling ratios for various detection molecules to the antibody, | ||
*3 to 8 biotin per IgG molecule (biotin MW: 244) | *3 to 8 biotin per IgG molecule (biotin MW: 244) | ||
| Line 16: | Line 17: | ||
*AF647: 3-7 moles of dye: mole of IgG | *AF647: 3-7 moles of dye: mole of IgG | ||
<biblio> | <biblio> | ||
#Paper1 pmid=9118596 | #Paper1 pmid=9118596 | ||
</biblio> | </biblio> | ||
Revision as of 10:29, 22 September 2008
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. Ideally the F:P ratio (fluorochrome : protein ratio) should be 1:1. 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. Below are some examples of suitable coupling ratios for various detection molecules to the antibody,
- 3 to 8 biotin per IgG molecule (biotin MW: 244)
- 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
