Are there any mutated IgG formats that FabRICATOR Xtra LALA cannot digest?
Yes, of the formats we have specifically tested, hIgG1-LALAPG (L234A, L235A, P329G), hIgG1-STR (L234S, L235T, G236R) and hIgG1-L234F, L235E, D265A could not be digested to satisfactory levels using FabRICATOR Xtra LALA. Please refer to footnote 4 in the instructions for use to determine whether your antibody is likely to be suitable for FabRICATOR Xtra LALA digestion.
Is there an enzyme for digestion of IgA2-only?
There is no known enzyme that targets IgA2-only since the amino acids in the hinge regions of IgA2 is present also in IgA1. However, IgASAP Sub1 will digest IgA1-only.
What enzyme should be selected for the digestion of IgA1?
IgASAP Sub1 specifically targets IgA1 and exhibits a faster reaction time (1 hour) compared to IgASAP Sub1+2 (16-18 hours). The cleavage site of IgASAP Sub1+2 is located six amino acids above that of IgASAP Sub1, resulting in the absence of potentially O-glycosylated residues on the Fab fragments. This characteristic can be advantageous in certain cases.
Can the IgASAP Sub1 digestion be performed at lower temperatures?
The optimal activity is at 37°C, but the digestion can also be performed at room temperature using a prolonged incubation time.
Which buffers are compatible with IgASAP Sub1?
The enzyme is active at pH 6.5 to 8.0, tolerates up to 300 mM NaCl, and does not require reducing conditions or co-factors for activity. PBS and TBS buffers are both compatible. Optimization may be required if buffers other than the recommended are used.
Does IgASAP Sub1 digest human IgA1-only?
IgASAP Sub1 may digest IgA1 from other primate species with similar hinge sequences, but this has not been tested. IgASAP Sub1 does not digest IgA2.
Does IgMBRAZOR digest only human IgM?
IgM from some species of monkey may also be digested by IgMBRAZOR.
Can IgMBRAZOR and FabRICATOR be used in the same reaction?
Yes, IgMBRAZOR and FabRICATOR can be used in combination.
Can IgMBRAZOR and PNGase F be used in the same reaction?
Yes, IgMBRAZOR and PNGase F can be used in combination if the reaction is performed under non-denaturing conditions. However, for complete deglycosylation of IgM using PNGase F denaturing conditions may be required and it is then recommended to perform the IgMBRAZOR digestion prior the PNGase F deglycosylation.
Is it possible to use IgMBRAZOR with very low concentrations of IgM?
It is possible to use IgMBRAZOR with low concentrations of IgM. However, this will lead to a decreased reaction rate, so for IgM concentrations below 0.5 mg/mL it is recommended to increase the amount of added enzyme and/or increase the incubation time. There is no risk of overdigestion and the incubation time can be prolonged to overnight if necessary.