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From 2010.igem.org

Andreas

IgG protease activity assay

Finished the repeated experiment and read the wavelength at 450 nm.

Blank: 80 μl PBS + 100 μl SureBlue™ peroxidase substrate + 100 μl 1 M HCl.

Sample loading
Ab dil.		1	2	3	4	5	6	7	8	9	10	11	12
1:500	A	Ind. IgGp A		Ind. IgGp B		Ind. SOD A		Ind. SOD B		Unind. IgGp A		Unind. IgGp B
1:250	B	Ind. IgGp A		Ind. IgGp B		Ind. SOD A		Ind. SOD B		Unind. IgGp A		Unind. IgGp B
–	C	PBS 1:500	PBS 1:250	Blank	–	–	–	–	–	–	–	–	–

Results

A450 measurements
	1	2	3	4	5	6	7	8	9	10	11	12
A	0.073	0.075	0.087	0.087	0.091	0.094	0.089	0.090	0.131	0.132	0.109	0.108
B	0.116	0.126	0.116	0.117	0.156	0.158	0.145	0.150	0.181	0.187	0.125	0.124
C	0.003	0.011	0.000	–	–	–	–	–	–	–	–	–

Unfortunately no pattern seen for the different samples. It seems like something else but the protein extracts is affecting proteolysis of the secondary antibodies. Maybe the primary antibodies conjugated to the agarose is not evenly distributed. Either way, it seems like the digestion is the result of proteolysis by E. coli proteases, not overexpressed IdeS.