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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.
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. Correspondingly, in 50 µl of serum you would
Q10  How should I store the FabRICATOR
F(ab’)2 Purification Kit?   		  A10 The FabRICATOR® Kit can be stored in its entirety at 4oC for up to 2 weeks. For longer storage times, please remove the lyophilized FabRICATOR Protease and store this component at -20°C, while keeping all other components at 4°C. It is important to note that the purification components may lose function, even after one freeze-thaw cycle.
Q11

Does FabRICATOR help with Mass Spec analysis?
  		  A11 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.
Q12  Where does FabRICATOR cleave?    A12 It cleaves between the 2 glycine residues just to the carboxyl-side of the disulfide bonds in the hinge region.
Q13  Does FabRICATOR prefer amino acid sequence for cleavage?   A13 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
Q14  Will FabRICATOR cleave my humanized IgG?     A14 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.
Q15  Will FabRICATOR cleave my Fc chimeric protein?     A15 Usually, FabRICATOR will efficiently cleave most Fc chimeras with hinge regions derived from human and rabbits, but not mouse.