Why Biolayer interferometry (BLI) is considered as a better instrument than surface plasmon resonance spectroscopy (SPR) in measuring label-free protein-ligand interactions?
In contrast to micro-fluidic SPR, which commonly delivers samples to a stationary sensor chip, BLI technology is established in an open shaking micro-well plate format without any micro-fluidics.
refractive index of the surrounding medium does not affect the interference pattern allows a variety of complex matrices
disposable biosensor tips, a range of standard surface chemistries is available
Dear Prof Jakob Wallner,
Thank you very much for your kind response. Yes, absence of micro-fluidics is a huge advantage for BLI (due to non-specific binding in control channel and blocking). However, I think BLI is limited for only His/GST or any other tagged proteins. Proteins like BSA and others can not be used to measure protein-ligand binding assay. Waiting for your expert comments on this point.
if you can tell me your protein-ligand of interest, I will try to recommend you an assay configuration.
Dear Prof Jakob Wallner,
I am trying to find the merits and demerits of BLI. I want to measure BSA (and His6 tagged protein)-ligand interactions.
Thank you
Debasis
Antibody-Specific Capture
• Anti-Human IgG Fc Capture (AHC)
Human IgG Fc region, kinetic analysis
• Anti-Human IgG Fc Capture (AHQ)
Human IgG Fc region, quantitation
• Anti-Mouse Fc Capture (AMC)
Mouse IgG1, 2a & 2b Fc regions, kinetic analysis
• Anti-Mouse Fc Capture (AMQ)
Mouse IgG1, 2a & 2b Fc regions, quantitation
• Anti-Human Fab-CHI (FAB2G)
Fab-CH1 domains of human IgG
• Protein A (ProA)
Quantitation of various species IgG
• Protein G (ProG)
Quantitation of various species IgG
• Protein L (ProL)
Quantitation of IgG via kappa light chain
Affinity Tag Capture
• Streptavidin (SA)
Biotinylated ligands
• High Precision Streptavidin (SAX)
Biotinylated ligands (4% CV)
• Super Streptavidin (SSA)
Biotinylated ligands (high-density surface, small molecules)
• Anti-GST (GST)
GST-tagged recombinant proteins
• Anti-Penta HIS (HIS1K)
HIS-tagged recombinant proteins
• Anti-Penta HIS 2nd Gen (HIS2)
HIS-tagged recombinant proteins
• Ni-NTA (NTA)
HIS-tagged recombinant proteins
Immobilization
• Amine Reactive 2nd Gen (AR2G)
Covalent coupling to reactive amine groups
• Aminopropylsilane (APS)
Adsorption to hydrophobic moieties
for your approach:
The easiest way:
immobilize your His6 tagged protein on follow sensor
Anti-Penta HIS (HIS1K)
HIS-tagged recombinant proteins
• Anti-Penta HIS 2nd Gen (HIS2)
HIS-tagged recombinant proteins
• Ni-NTA (NTA)
HIS-tagged recombinant proteins
Immobilization
you can switch the orientation, use
Streptavidin (SA) Sensor: biotinylate BSA
or you can immobilize BSA on
• Amine Reactive 2nd Gen (AR2G)
Covalent coupling to reactive amine groups
• Aminopropylsilane (APS)
Adsorption to hydrophobic moieties
Thank you very much for your valuable information. Its really useful for us. I know you are an user of BLI instrument (for protein-liposome interaction), but have used this instrument to measure protein-small molecule interactions? If yes, is there any limit for molecular weight ratio between protein and small molecules (upper and lower limit)?
In my case I have an enzyme of 47 kDa and ligand of 300 Da. Do you think, i can measure their interaction by BLI?
Thank you again for your useful information about BLI instrument.
it depends on the device
The OCTET QK has dynamic range for quantitation of proteins and peptides down to
> 5 kDa
The Octet RED > 100 Dalton
you can also use the OCTET QK, immobilize the 300 Da ligand, because the signal for immobilization step does not matter
K2 has the same sensitivity as the RED (100 Dalton) but you can measure only two Sensor parallel instead 8 Sensors
BLI instrument shows much less sensitivity than SPR instrument. Issues of repeatability also there with BLI
Hi,
I have a protein whic is His-tagged and I want to use BLI to measure its interaction with a small molecule. Is it suitable to use the Ni-NTA sensor to measure the interaction between the recombinant potein and a small molecule? or is it only suitable to measure protein-potein interaction using this kind of sensors?
In my impression, the major advantage of BLI is easy-to-use and a little bit higher throughput. As to the sensitivity, we have determined the binding kinetics of 124Da pyrazinoic acid on the pyrazinamide-target protein, PanD.
Sun Q, Li X, Perez LM, Shi W, Zhang Y, Sacchettini JC. The molecular basis of pyrazinamide activity on Mycobacterium tuberculosis PanD. Nat Commun. 2020 Jan;11(1):339
I also made an R package to implement our data analysis routine. It's in the CRAN repository, and free to download and run on your dataset now.
Sun Q, Li X, Sacchettini JC (2020). smoke: Small Molecule Octet/BLI Kinetics Experiment. R package version 2.0.0. https://CRAN.R-project.org/package=smoke
Both organic solvent (DMSO) and higher salt concentration affect SPR measurements.
Recently I tried to bind a histagged protein onto the Octet Ni-NTA sensor and I got a negative response. This is the first time I have observed this phenomenon with any protein. I performed buffer exchange so the buffer is the same as baseline, but I still got the same result. Has anyone else encountered this problem?
Hi William Sun ,
We recently tried to load a virion onto a biosensor and we also obtained a negative loading signal. Is it possible that the octet device has a maximum "size" threshold that eventually disrupt the interferometry pattern ?
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