binding-characterization

Name: binding-characterization
Author: adaptyvbio

35installs20forks

How to install binding-characterization?

adaptyvbio/protein-design-skills/binding-characterization

$npx -y skills add adaptyvbio/protein-design-skills --skill binding-characterization

Installs into the current project.

Summary

Guidance for SPR and BLI binding characterization experiments. Use when: (1) Planning binding kinetics experiments, (2) Troubleshooting poor/no binding signal, (3) Interpreting kinetic data artifacts, (4) Choosing between SPR vs BLI platforms.

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SKILL.md

1	# Binding Characterization: SPR and BLI
2
3	## SPR vs BLI Decision Matrix
4
5	\| Factor \| Choose SPR \| Choose BLI \|
6	\|--------\|------------\|------------\|
7	\| Sensitivity \| Small molecules, fragments (<500 Da) \| Large complexes, antibodies \|
8	\| Throughput \| Low-medium (serial) \| High (96-well parallel) \|
9	\| Sample purity \| Required (clogs fluidics) \| Tolerates crude lysates \|
10	\| Kinetic resolution \| Higher (better for fast kinetics) \| Lower \|
11	\| Mass transport \| More sensitive (may distort kon) \| Less sensitive \|
12	\| Maintenance \| High (fluidics system) \| Low (dip-and-read) \|
13	\| Sample consumption \| Higher (continuous flow) \| Lower \|
14	\| Cost per experiment \| Lower chip cost, higher run cost \| Higher tip cost, lower run cost \|
15
16	## Key differences
17
18	### SPR (Surface Plasmon Resonance)
19	- Mechanism: Detects refractive index changes at gold surface
20	- Surface: Gold chip with dextran matrix (CM5, CM7, etc.)
21	- Flow: Continuous microfluidics
22	- Best for: Small molecules, high-affinity, precise kon/koff
23
24	### BLI (Biolayer Interferometry)
25	- Mechanism: Measures optical interference pattern shift
26	- Surface: Fiber optic biosensor tips (SA, Ni-NTA, AHC)
27	- Flow: Dip-and-read (no microfluidics)
28	- Best for: High-throughput, crude samples, antibody screening
29
30	---
31
32	## Troubleshooting: Why BLI works but SPR doesn't
33
34	\| Cause \| Mechanism \| Solution \|
35	\|-------\|-----------\|----------\|
36	\| Hydrophobic CDRs \| Adsorb to SPR gold/dextran surface \| Add 0.05% Tween-20, use CM7 chip with longer dextran \|
37	\| Aggregation \| Mass transport artifacts in SPR fluidics \| Filter sample (0.22μm), reduce ligand density \|
38	\| High instability \| Degrades during continuous flow \| Shorter cycle time, add stabilizers (trehalose 5%) \|
39	\| Charge mismatch \| Nonspecific binding to charged dextran \| Adjust buffer pH ±1 from pI, add BSA 1mg/mL \|
40	\| Slow dissociation \| Long regeneration needed (damages ligand) \| Use BLI (disposable tips) \|
41
42	### Why SPR works but BLI doesn't
43
44	\| Cause \| Mechanism \| Solution \|
45	\|-------\|-----------\|----------\|
46	\| Small analyte \| BLI less sensitive for <10 kDa \| Use SPR with appropriate chip \|
47	\| Weak affinity (KD >10μM) \| Fast dissociation in BLI dip \| Increase analyte concentration \|
48	\| Low expression \| Not enough signal \| Increase biosensor loading \|
49
50	---
51
52	## Mass transport considerations
53
54	Mass transport limitation occurs when analyte cannot diffuse to the surface fast enough to maintain equilibrium. This distorts kinetic parameters.
55
56	### Symptoms
57	- Observed kon appears slower than true kon
58	- Linear association phase (instead of exponential)
59	- kon varies with ligand density
60	- Rmax varies with flow rate
61
62	### When mass transport matters
63	- High-affinity interactions (kon >10^6 M^-1s^-1)
64	- High ligand density (>500 RU)
65	- Slow flow rates (<30 μL/min in SPR)
66	- Large analytes (slow diffusion)
67
68	### Mitigation strategies
69
70	\| Strategy \| SPR \| BLI \|
71	\|----------\|-----\|-----\|
72	\| Reduce ligand density \| <200 RU for high-affinity \| <0.5 nm shift loading \|
73	\| Increase flow rate \| 50-100 μL/min \| Increase shake speed (1000 rpm) \|
74	\| Use oriented immobilization \| His-tag capture \| Biotinylated ligand \|
75	\| Include in fitting \| Mass transport model (kt) \| Usually less critical \|
76
77	---
78
79	## Nonspecific binding mitigation
80
81	### Buffer additives (ranked by effectiveness)
82
83	\| Additive \| Concentration \| Mechanism \| Best For \|
84	\|----------\|---------------\|-----------\|----------\|
85	\| BSA \| 0.5-1 mg/mL \| Blocks hydrophobic sites \| General use \|
86	\| Tween-20 \| 0.02-0.05% \| Prevents surface adsorption \| Hydrophobic analytes \|
87	\| Trehalose \| 1-5% \| Stabilizes + blocks \| Unstable proteins \|
88	\| Sucrose \| 5% \| BLI-specific blocker \| BLI tips \|
89	\| Carboxymethyl dextran \| 1 mg/mL \| Competitive blocking \| SPR with charged proteins \|
90	\| NaCl \| 150-500 mM \| Reduces ionic interactions \| Charged proteins \|
91
92	### pH optimization
93	- Keep buffer pH at least 1 unit away from analyte pI
94	- pI near 7: Use pH 6.0 or 8.0 buffer
95	- Acidic proteins (pI <5): Use neutral or basic buffer
96	- Basic proteins (pI >9): Use slightly acidic buffer
97
98	### Reference subtraction
99	Always include:
100	- Blank reference channel (no ligand)
101	- Buffer-only injections
102	- Non-specific binding controls
103
104	---
105
106	## Regeneration conditions
107
108	### SPR regeneration scouting (try in order)
109
110	\| Condition \| Targets \| Caution \|
111	\|-----------\|---------\|---------\|
112	\| 10 mM Glycine pH 2.0-2.5 \| Most protein-protein \| May denature ligand \|
113	\| 10 mM Glycine pH 1.5 \| Strong interactions \| Harsh, limit exposure \|
114	\| 1-2 M NaCl \| Ionic interactions \| Mild, try first \|
115	\| 10 mM NaOH \| Very stable lig

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binding-characterization

35installs20forks

View on GitHub

How to install binding-characterization?

adaptyvbio/protein-design-skills/binding-characterization

$npx -y skills add adaptyvbio/protein-design-skills --skill binding-characterization

Installs into the current project.

Summary

Files · 1

View on GitHub

SKILL.md

1	# Binding Characterization: SPR and BLI
2
3	## SPR vs BLI Decision Matrix
4
5	\| Factor \| Choose SPR \| Choose BLI \|
6	\|--------\|------------\|------------\|
7	\| Sensitivity \| Small molecules, fragments (<500 Da) \| Large complexes, antibodies \|
8	\| Throughput \| Low-medium (serial) \| High (96-well parallel) \|
9	\| Sample purity \| Required (clogs fluidics) \| Tolerates crude lysates \|
10	\| Kinetic resolution \| Higher (better for fast kinetics) \| Lower \|
11	\| Mass transport \| More sensitive (may distort kon) \| Less sensitive \|
12	\| Maintenance \| High (fluidics system) \| Low (dip-and-read) \|
13	\| Sample consumption \| Higher (continuous flow) \| Lower \|
14	\| Cost per experiment \| Lower chip cost, higher run cost \| Higher tip cost, lower run cost \|
15
16	## Key differences
17
18	### SPR (Surface Plasmon Resonance)
19	- Mechanism: Detects refractive index changes at gold surface
20	- Surface: Gold chip with dextran matrix (CM5, CM7, etc.)
21	- Flow: Continuous microfluidics
22	- Best for: Small molecules, high-affinity, precise kon/koff
23
24	### BLI (Biolayer Interferometry)
25	- Mechanism: Measures optical interference pattern shift
26	- Surface: Fiber optic biosensor tips (SA, Ni-NTA, AHC)
27	- Flow: Dip-and-read (no microfluidics)
28	- Best for: High-throughput, crude samples, antibody screening
29
30	---
31
32	## Troubleshooting: Why BLI works but SPR doesn't
33
34	\| Cause \| Mechanism \| Solution \|
35	\|-------\|-----------\|----------\|
36	\| Hydrophobic CDRs \| Adsorb to SPR gold/dextran surface \| Add 0.05% Tween-20, use CM7 chip with longer dextran \|
37	\| Aggregation \| Mass transport artifacts in SPR fluidics \| Filter sample (0.22μm), reduce ligand density \|
38	\| High instability \| Degrades during continuous flow \| Shorter cycle time, add stabilizers (trehalose 5%) \|
39	\| Charge mismatch \| Nonspecific binding to charged dextran \| Adjust buffer pH ±1 from pI, add BSA 1mg/mL \|
40	\| Slow dissociation \| Long regeneration needed (damages ligand) \| Use BLI (disposable tips) \|
41
42	### Why SPR works but BLI doesn't
43
44	\| Cause \| Mechanism \| Solution \|
45	\|-------\|-----------\|----------\|
46	\| Small analyte \| BLI less sensitive for <10 kDa \| Use SPR with appropriate chip \|
47	\| Weak affinity (KD >10μM) \| Fast dissociation in BLI dip \| Increase analyte concentration \|
48	\| Low expression \| Not enough signal \| Increase biosensor loading \|
49
50	---
51
52	## Mass transport considerations
53
54	Mass transport limitation occurs when analyte cannot diffuse to the surface fast enough to maintain equilibrium. This distorts kinetic parameters.
55
56	### Symptoms
57	- Observed kon appears slower than true kon
58	- Linear association phase (instead of exponential)
59	- kon varies with ligand density
60	- Rmax varies with flow rate
61
62	### When mass transport matters
63	- High-affinity interactions (kon >10^6 M^-1s^-1)
64	- High ligand density (>500 RU)
65	- Slow flow rates (<30 μL/min in SPR)
66	- Large analytes (slow diffusion)
67
68	### Mitigation strategies
69
70	\| Strategy \| SPR \| BLI \|
71	\|----------\|-----\|-----\|
72	\| Reduce ligand density \| <200 RU for high-affinity \| <0.5 nm shift loading \|
73	\| Increase flow rate \| 50-100 μL/min \| Increase shake speed (1000 rpm) \|
74	\| Use oriented immobilization \| His-tag capture \| Biotinylated ligand \|
75	\| Include in fitting \| Mass transport model (kt) \| Usually less critical \|
76
77	---
78
79	## Nonspecific binding mitigation
80
81	### Buffer additives (ranked by effectiveness)
82
83	\| Additive \| Concentration \| Mechanism \| Best For \|
84	\|----------\|---------------\|-----------\|----------\|
85	\| BSA \| 0.5-1 mg/mL \| Blocks hydrophobic sites \| General use \|
86	\| Tween-20 \| 0.02-0.05% \| Prevents surface adsorption \| Hydrophobic analytes \|
87	\| Trehalose \| 1-5% \| Stabilizes + blocks \| Unstable proteins \|
88	\| Sucrose \| 5% \| BLI-specific blocker \| BLI tips \|
89	\| Carboxymethyl dextran \| 1 mg/mL \| Competitive blocking \| SPR with charged proteins \|
90	\| NaCl \| 150-500 mM \| Reduces ionic interactions \| Charged proteins \|
91
92	### pH optimization
93	- Keep buffer pH at least 1 unit away from analyte pI
94	- pI near 7: Use pH 6.0 or 8.0 buffer
95	- Acidic proteins (pI <5): Use neutral or basic buffer
96	- Basic proteins (pI >9): Use slightly acidic buffer
97
98	### Reference subtraction
99	Always include:
100	- Blank reference channel (no ligand)
101	- Buffer-only injections
102	- Non-specific binding controls
103
104	---
105
106	## Regeneration conditions
107
108	### SPR regeneration scouting (try in order)
109
110	\| Condition \| Targets \| Caution \|
111	\|-----------\|---------\|---------\|
112	\| 10 mM Glycine pH 2.0-2.5 \| Most protein-protein \| May denature ligand \|
113	\| 10 mM Glycine pH 1.5 \| Strong interactions \| Harsh, limit exposure \|
114	\| 1-2 M NaCl \| Ionic interactions \| Mild, try first \|
115	\| 10 mM NaOH \| Very stable lig

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