Table 1.

Typical set up of the binding competition assay in a 96-well Microplate for measuring peptide binding to MHCII molecules

Target peptide (nM)a		Target peptide 1			Target peptide 2			Target peptide 3			Target peptide 4
		+ MHCII +Alexa488-FRR-HA306-318			+ MHCII +Alexa488-FRR-HA306-318			+ MHCII +Alexa488-FRR-HA306-318			+ MHCII +Alexa488-FRR-HA306-318
		1	2	3	4	5	6	7	8	9	10	11	12
20000	A
4000	B
800	C
160	D
32	E
6.4	F
1.28	G
0	H	FP_no_compb						FP_freec

^aTarget peptide concentrations starts at 20000 nM with a dilution factor of 5. Assay for each peptide is performed with three replicates. The last row H has no competing target peptide.

^bRow H, columns 1-6 contain only MHCII and labeled probe peptide, no competing target peptide, allowing for measurement of FP for labeled probe peptide bound with MHCII without competitor peptide (FP_no_comp).

^cRow H, columns 7-12 contain only labeled probe peptide, no MHCII and competing target peptide, allowing for measurement of FP for free unbound labeled probe peptide (FP_free).