rProtein A Sepharose Fast Flow
rProtein A Sepharose 4 Fast Flow is an affinity resin for antibody purification at laboratory and process scale.
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Protein G is a good first choice for general purpose capture of antibodies at laboratory scale because it binds a broader range of IgG from eukaryotic species and also binds to more subclasses of IgG. The table below shows the relative binding strengths of antibodies from various species to protein G and protein A.
Table 3.1 from Antibody Purification Handbook. Relative binding strengths of antibodies from various species to protein G and protein A as measured in a competitive ELISA test. The amount of IgG required to give a 50% inhibition of binding of rabbit IgG conjugated with alkaline phosphatase was determined.
Species | Subclass | Protein G binding | Protein A binding |
---|---|---|---|
Human | IgA | — | variable |
IgD | — | — | |
IgE | — | — | |
IgG1 | ++++ | ++++ | |
IgG2 | ++++ | ++++ | |
IgG3 | ++++ | — | |
IgG4 | ++++ | ++++ | |
IgM* | — | variable | |
Avian egg yolk | IgY† | — | — |
Cow | ++++ | ++ | |
Dog | + | ++ | |
Goat | ++ | — | |
Guinea pig | IgG1 | ++ | ++++ |
Hamster | ++ | + | |
Horse | ++++ | ++ | |
Species | Subclass | Protein G binding | Protein A binding |
Koala | + | — | |
Llama | + | — | |
Monkey (rhesus) | ++++ | ++++ | |
Mouse | IgG1 | ++++ | + |
IgG2a | ++++ | ++++ | |
IgG2 | +++ | +++ | |
IgG3 | +++ | ++ | |
IgM* | — | variable | |
Pig | +++ | +++ | |
Rabbit | +++ | ++++ | |
Rat | IgG1 | + | — |
IgG2a | ++++ | — | |
IgG2b | ++ | — | |
IgG3 | ++ | + | |
Sheep | ++ | +/— |
* Purified using HiTrap IgM Purification HP columns.
† Purified using HiTrap IgY Purification HP columns.
++++ = strong binding.
++ = medium binding.
— = weak or no binding
Use the immunoprecipitation protocol found in the Instructions for the Immunoprecipitation Starter Pack (literature code 71-5017-54).
After binding of the antibody to the beads, perform cross-linking according to the following protocol (batch protocol):
Binding buffer: TBS (50 mM Tris, 150 mM NaCl, pH 7.5)
Wash buffer: TBS with 2 M urea, pH 7.5
Elution buffer: 0.1 M glycine with 2 M urea, pH 2.9
Cross-link solutions:
- 200 mM triethanolamine, pH 8.9
- 50 mM DMP (Dimethyl pimelimidate dihydrochloride) in 200 mM triethanolamine, pH 8.9
- 100 mM ethanolamine, pH 8.9
Wash the beads with 4 gel volumes of binding buffer - centrifuge for 20 s at 12 000 x g
Change buffer: Add 4 gel volumes of triethanolamine and centrifuge for 20 s at 12 000 x g
Cross-link : Add 4 gel volumes of DMP in triethanolamine. Fully suspend the gel by manual inversion and incubate with slow, end-over-end mixing for 60 min. Centrifuge for 20 s at 12 000 x g.
Wash: Add 4 gel volumes of triethanolamine and mix by manual inversion. Centrifuge for 20 s at 12 000 x
Block: Add 4 gel volumes of ethanolamine. Mix by manual inversion and incubate end-over -end for 15 min. Centrifuge for 20 s at 12 000 x g
Remove unbound antibody: Add 4 gel volumes of elution buffer and centrifuge for 20 s at 12 000 x g.
Wash: Add 4 gel volumes of binding buffer and centrifuge for 20 seconds at 12 000 x g. Perform this step 2 times total.
See the table below for differences between the two products.
Products | nProtein A Sepharose 4 Fast Flow | rProtein A Sepharose Fast Flow |
---|---|---|
Product codes | 17-5280-xx | 17-1279-xx |
Ligand | n (native) Protein A | r (recombinant) Protein A |
Produced in strain | S. aureus | E. coli |
Coupling chemistry | Cyanogen bromide activation | Epoxy activation |
Binding capacity (mg human IgG/ml) | > 35 | 50 |
pH stability | 3-9 (working) 2-10 (short term) |
2-9 (working) 2-11 (short term) |
Common problems with, and general tips for immunoprecipitation can be found in the Immunoprecipitation Starter Pack Instructions (literature code 71-5017-54).