Table 2.

Binding affinities of 9 selected hs2dAb fused to a 6HIS tag measured by surface plasmon resonance single cycle kinetics method. Dissociation equilibrium constant KD corresponds to the ratio between off-rate and on-rate kinetic constant K_off/K_on. Non relevant hs2dAb were used as negative controls and gave no detectable binding signal. A positive control endowed with subnanomolar affinity, the GFP binder VHH-GFP4, was analyzed in parallel to the GFP hs2dAbs. A KD of 1.55^–10 M was measured for VHH-GFP4 which is similar to published values. The binding properties of the conformational H12 hs2dAb to the GTP loaded RHOA subfamily were measured using the L63 or L61 constitutively active mutants of RHO, RHOB, RHOC, RAC1 and CDC42 related small GTPases, as well as the negative mutant T19N of RHOA. ('no' means no detectable binding). 

DOI: http://dx.doi.org/10.7554/eLife.16228.023

hs2dAb-6xHis	Antigen	kon (M−1 s−1)	koff (s−1)	KD(M)
R2TB5 anti-GFP	GFP	1.24 10+6	3.05 10−4	2.45 10−10
R3SD1 anti-GFP	GFP	7.07 10+5	6.68 10−4	9.44 10−10
R3SE4 anti-GFP	GFP	1.45 10+5	5.83 10−4	4.01 10−9
Llama VHH GFP4	GFP	2.99 10+5	4.65 10−5	1.55 10−10
D4 anti-Her2	Her2	1.79 10+5	1.63 10−4	9.11 10−10
A10 anti-Her2	Her2	1.66 10+4	4.88 10−5	2.94 10−9
B9 anti-Cherry	mCherry	6.14 10+4	1.68 10−4	2.74 10−9
E4 anti-Cherry	mCherry	6.57 10+4	2.03 10−4	3.10 10−9
B3 anti-Cherry	mCherry	6.19 10+4	2.71 10−4	4.38 10−9
H12 anti-RHO.GTP	RHOA Q63L	4.81 10+5	1.28−4	2.65 10−10
H12 anti-RHO.GTP	RHOB Q63L	2.24 10+5	3.59−4	1.57 10−9
H12 anti-RHO.GTP	RHOC Q63L	1.12 10+6	5.41−5	4.79 10−11
H12 anti-RHO.GTP	RHOA T19N	no	no	no
H12 anti-RHO.GTP	RAC1 Q61L	7.53 10+5	2.55−4	3.3 10−10
H12 anti-RHO.GTP	CDC42 Q61L	no	no	no