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FAQ - FabRICATOR® IgG Protease
Questions |
Answers | ||||
| Q1 |
Which IgG does FabRICATOR® cleave? | A1 | FabRICATOR®cleaves all subclasses of human, rabbit, monkey and sheep IgG and one subclass of mouse IgG -- IgG3 but not mouse IgG1, IgG2a and IgG2b. | ||
| Q2 | Where does FabRICATOR® cleave the IgG molecule? |
A2 | FabRICATOR® cleave the IgG molecule in a single, specific, site below the hinge region of the anitbody leaving a F(ab')2 fragment and a Fc-fragment. | ||
| Q3 | Is it possible to get a F(ab')1 fragments from F(ab')2 fragments? |
A3 | Yes, after FabRICATOR cleavage, use 2-MEA (2-mercapto ethylamine) at a concentration of 50 mM, to break disulfide bond. Click here to see the protocol. | ||
| Q4 | How much Reagent 2 particles should I use for each µg of digested IgG? | A4 | The Reagent 2 concentration in the FabRICATOR® Test Kit (50 u) is lower than in the FabRICATOR® Kit (2000 and 5000 u). If you are using Reagent 2 particles from the FabRICATOR® Test Kit you should use 1 µl/ µg digested IgG and if you are using the Reagent 2 particles from FabRICATOR® Kit you should use 0.3 µl/ µg digested IgG. | ||
| Q5 | Is it possible to use FabRICATOR® in a buffer at another pH then 6.6? | A5 | Yes, but the reaction time should be increased. Best activity is obtained at pH 6.6 and 37ºC. . | ||
| Q6 | Is FabRICATOR® active at room temperature | A6 | Yes, but the reaction time should be increased by 15 min. | ||
| Q7 | Is it possible to use FabRICATOR® in a buffer containing EDTA or HSA? | A7 | Yes. | ||
| Q8 | Do you have any data regarding cleavage if IgG in human serum? | A8 | FabRICATOR® cleaves well in serum. | ||
| Q9 | How much FabRICATOR® do you need to cleave IgG in 50 µl of normal human serum? | A9 | If we assume that the antibody concentration in serum is 15 mg/ml you would have to add 10,000 – 15,000 units to a 1 ml sample of serum. | ||
| Q10 |
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A10 | Yes, because FabRICATOR protease cleaves at a single amino acid location with no degradation products, it produces pristine F(ab’)2 and Fc fragments which can be directly analyzed by mass spec or X-crystallography. | ||
| Q11 | Where does FabRICATOR cleave? | A11 | It cleaves between the 2 glycine residues just to the carboxyl-side of the disulfide bonds in the hinge region. | ||
| Q12 | Does FabRICATOR prefer amino acid sequence for cleavage? | A12 | Yes, FabRICATOR will efficiently cleave between the glycine residues in the following conserved sequence (where the 2 cysteine create the thiol bridges): D-K-T-H-T-C-P-P-C-P-A-P-E-L-L-G-G-P-S-V | ||
| Q13 | Will FabRICATOR cleave my humanized IgG? | A13 | Usually, FabRICATOR will efficiently cleave just about any IgG with the conserved sequence above, or hinge regions that have close homology to the human sequence. | ||
| Q14 | Will FabRICATOR cleave my Fc chimeric protein? | A14 | Usually, FabRICATOR will efficiently cleave most Fc chimeras with hinge regions derived from human and rabbits, but not mouse. | ||
| Q15 | How do I elute the Fc fragment from my FragIT Purfication column (microspin)? | A15 | Fc fragments can be eluted using 200µL of a 0.1M glycine buffer pH=3. Repeat the elution two additional times for maximum recovery. | ||
| Q15 | How long is reconstituted FabRICATOR stable for? | A15 | 3 months at -20ºC and 1 month at 4ºC |