Digestion of IgM in Serum using IgMBRAZOR™

Application

Isotype-specific digestion of IgM and IgG in serum, allowing simultaneous analysis without enzymatic cross-interference.

IgM constitutes around 5% of the total amount of immunoglobulins in normal human serum, and the ability to selectively digest IgM can be of great value in research and diagnostics as well as in bioprocessing and therapeutic applications.

The specificity of IgMBRAZOR makes it an excellent tool for such isotype-specific digestion of IgM in human serum samples. Here, human serum was incubated with IgMBRAZOR Lyophilized and/or FabRICATOR Lyophilized for 30 minutes at 37°C before analysis with SDS-PAGE and western blot.

Efficient digestion of IgM in human serum

IgM was successfully digested using IgMBRAZOR and IgG using FabRICATOR. Both enzymes were isotype-specific, and no interference with the FabRICATOR activity was observed when combining the two enzymes.

Isotype-specific digestion of IgM with no interference when combined with FabRICATOR

Activity of IgMBRAZOR in human serum. a) IgMBRAZOR Lyophilized was added to human serum and incubated for 30 minutes at 37°C. The digested sample and control samples of human serum, human IgM, and human IgG were separated on two reducing SDS-PAGE gels. One of the gels was stained, and the other transferred to a membrane. After blocking of the membrane in a casein solution, alkaline phosphatase conjugated α-human IgM HC antibody was added and incubated for 1 h at room temperature. The membrane was thoroughly washed before addition of the chromogenic substrate BCIP/NBT. b) IgMBRAZOR Lyophilized and FabRICATOR Lyophilized were incubated in human serum for 30 minutes at 37°C, both individually and in combination. The digested samples and controls were prepared as in (a), except for the primary antibody solution used; alkaline phosphatase conjugated α-human IgG HC. Black arrows indicate the expected molecular weight of IgM HC that due to the low concentration is not visible in the serum samples.

Related Products

Resources