Homogenous pool of Fab fragments from IgG in two hours

A beautiful way to circumvent the micro heterogeneity and boost the low yield that is often the result of fragmentation using other proteolytic enzymes like pepsin or papain is to combine our FabRICATOR™ enzyme with 2-MEA (2-mercaptoethanolamine or Cysteamine). This procedure is simple and effective with excellent yield. Also, it can be completed in two hours!

 

For the researchers who wish to do bioconjugation of the generated Fab fragments to other biomolecules or surfaces there’s good news: The generated Fab fragments contains the hinge thiols available for conjugation through standard maleimide chemistry. You don’t even need to remove the Fc fragments before conjugation, just perform a simple desalting of the fragments and then proceed directly with your conjugation reaction. We will publish a protocol for this shortly.

Use FabRICATOR™ or FragIT™ to cut the IgG just below the hinge region. This procedure takes approximately 30 minutes. You will end up with a homogenous pool of F(ab')2 fragments and Fc fragments. Reaction should be performed in a buffer conatining EDTA,, for example, 50 mM Phosphate, 150 mM NaCl, 5 mM EDTA, pH 7.2.
Next, add 2-MEA (2-mercaptoethanol amine) to a final concentration of 50 mM and incubate at 37ºC for 90 minutes. There is no need to remove the FabRICATOR™ enzyme if this is present. (Make a stock solution of 2-MEA in same buffer as above)
Now your solution conatins Fab fragments, Fc fragments, 2-MEA and possibly FabRICATOR enzyme. To remove the 2-MEA a simple desalting column can be used. All major brands of desalting columns will work (for example GE (NAP 5, NAP 10, NAP 25) or other G25 columns). Volumes will vary depending on fragmentation tool chosen.