What are the contributions in this paper?

Nanobodies have been used as chaperones for crystallizing complex biological systems such as membrane proteins, transient multiprotein assemblies, and intrinsically disordered proteins this paper.

How many bp of cDNA should be amplified?

Two DNA fragments should be amplified: fragments of ~700 bp representing the heavy chain–only antibody repertoire and fragments of 1,000 bp corresponding to the heavy chain of the conventional antibodies (supplementary Fig. 1a).

How do you remove the phage particles from the wells?

After each wash step, remove excess liquid by tapping the emptied plates once on a paper towel, avoiding cross-contamination of the phage to the neighboring wells.

How many dN6 random primers are stored in a sigma-a?

These customsynthesized primers (Sigma-Aldrich) are stored at −20 °C for years as stock solutions at 100 µM in 10 mM Tris-HCl buffer (pH 8.5) dN6 random primers (5′-NNNNNN-3′) are custom-synthesized and stored at −20° for years as a stock solution in 10 mM Tris-HCl buffer (pH 8.5) at a 2.5 µg µl−1 concentration EQUIPMENT

What is the robust technique for phage display?

Phage display is certainly the most robust technique, but yeast display53,54 or bacterial display55 can also be used to select Nanobodies from immune libraries.

(Open Access) A general protocol for the generation of Nanobodies for structural biology (2014) | Els Pardon

Vrije Universiteit Brussel

A general protocol for the generation of Nanobodies for structural Biology

Pardon, Els; Laeremans, Toon; Triest, Sarah; Rasmussen, Soren G. F.; Ruf, Armin;

Muyldermans, Serge; Hol, Wim G. J.; Kobilka, Brian K; Steyaert, Jan

Published in:

Nature Protocols

DOI:

10.1038/nprot.2014.039

Publication date:

2014

Document Version:

Final published version

Link to publication

Citation for published version (APA):

Pardon, E., Laeremans, T., Triest, S., Rasmussen, S. G. F., Ruf, A., Muyldermans, S., ... Steyaert, J. (2014). A

general protocol for the generation of Nanobodies for structural Biology. Nature Protocols, 9, 674-693.

https://doi.org/10.1038/nprot.2014.039

No part of this publication may be reproduced or transmitted in any form, without the prior written permission of the author(s) or other rights

holders to whom publication rights have been transferred, unless permitted by a license attached to the publication (a Creative Commons

license or other), or unless exceptions to copyright law apply.

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Download date: 09. Aug. 2022

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NATURE PROTOCOLS

INTRODUCTION

The production of diffraction-quality crystals remains the major

bottleneck in macromolecular X-ray crystallography. Collective

efforts of several laboratories have demonstrated that Nanobodies

are exquisite chaperones for crystallizing complex biological

systems such as membrane proteins

1–3

, transient multiprotein

assemblies

2,4–6

, transient conformational states

and intrinsically

disordered proteins

7,8

. Further, they can be used as structural

probes of protein misfolding and fibril formation

9,10

. Nanobodies

are the small (15 kDa) and stable single-domain fragments

harboring the full antigen-binding capacity of the original

heavy chain–only antibodies that naturally occur in camelids

11,12

Nanobodies are encoded by single gene fragments, they are

easily produced in microorganisms and they exhibit a superior

stability compared with derivatives of conventional antibodies

such as Fabs or scFvs. Because of their compact prolate shape,

Nanobodies expose a convex paratope and have access to

cavities or clefts on the surface of proteins

1,13,14

that are often

inaccessible to conventional antibodies. These cryptic epitopes

can be readily recognized by the long CDR3 loop of the Nanobody.

In our experience, Nanobodies raised in vivo by immunization

against, and selected on, properly folded proteins systematically

recognize discontinuous amino acid segments of the native

protein conformation (i.e., conformational epitopes), making

them ideal tools to selectively stabilize specific conformational

states of (membrane) proteins.

For the discovery of Nanobodies as crystallization chaper-

ones, ~1 mg of functional protein is required. The generation of

in vivo–matured Nanobodies can therefore be incorporated in

the crystallization pipeline even before the purification of the

protein has been fully optimized and scaled up. Nanobodies

binding conformational epitopes (conformational Nanobodies)

can subsequently be used for preparing pure, homogeneous and

highly concentrated monodisperse samples that are required for

crystallization

. If no native purified protein is available, genetic

and cell-based vaccinations, combined with cell-based selection

approaches, have been successfully applied in our laboratory and

elsewhere to generate Nanobodies against target proteins in their

native conformation

16–18

Comparisons with other approaches

Here we present a general protocol for the generation, selection

and purification of recombinant in vivo–matured Nanobodies

for structural biology

1–5,7,9,19–30

that takes 3–4 months. Our

Nanobody discovery platform has the competitive advantage over

other recombinant crystallization chaperones

31–33

that the cloned

Nanobody library represents the full collection of the naturally cir-

culating, humoral antigen-binding repertoire of heavy chain–only

antibodies, in contrast to combinatorial libraries of conventional

antibody fragments. Because Nanobodies are encoded by single

exons, the full antigen-binding capacity of in vivo–matured anti-

bodies can be cloned and efficiently screened for high-affinity

binders, allowing one to fully exploit the humoral response of large

mammals against native antigens. To our knowledge, there are no

indications that in vivo–matured Nanobodies induce non-native

conformations. Surely, immature B cells expressing antibodies

that have to pay a substantial energetic penalty for distorting the

antigen structure will have a lower probability of proliferation and

differentiation into mature antibody-secreting B lymphocytes.

Limitations

With nearly 20 years of experience we have learned that conforma-

tional Nanobodies can be identified against any properly folded

protein. In those cases in which we failed in a first attempt, we

successfully performed new immunizations or pannings, paying

special attention to the quality of the antigen, thereby learning

that good protein biochemistry is the key to success. Although

Nanobodies are good at binding conformational epitopes on

folded proteins with high affinity, they perform poorly at binding

A general protocol for the generation of Nanobodies

for structural biology

Els Pardon

1,2

, Toon Laeremans

1,2

, Sarah Triest

1,2

, Søren G F Rasmussen

, Alexandre Wohlkönig

1,2

, Armin Ruf

Serge Muyldermans

2,5

, Wim G J Hol

, Brian K Kobilka

& Jan Steyaert

1,2

Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

Structural Biology Research Center, Vlaams Instituut voor Biotechnologie (VIB),

Brussels, Belgium.

Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.

Pharma Research

and Early Development (pRED), Small Molecule Research, Discovery Technologies, F. Hoffmann-La Roche, Basel, Switzerland.

Cellular and Molecular Immunology,

VUB, Brussels, Belgium.

Department of Biochemistry, Biomolecular Structure Center, School of Medicine, University of Washington, Seattle, Washington, USA.

Department of Molecular and Cellular Physiology, School of Medicine, Stanford University, Stanford, California, USA. Correspondence should be addressed

to J.S. (jan.steyaert@vib-vub.be).

Published online 27 February 2014; doi:10.1038/nprot.2014.039

There is growing interest in using antibodies as auxiliary tools to crystallize proteins. Here we describe a general protocol

for the generation of Nanobodies to be used as crystallization chaperones for the structural investigation of diverse

conformational states of flexible (membrane) proteins and complexes thereof. Our technology has a competitive advantage

over other recombinant crystallization chaperones in that we fully exploit the natural humoral response against native antigens.

Accordingly, we provide detailed protocols for the immunization with native proteins and for the selection by phage display

of in vivo–matured Nanobodies that bind conformational epitopes of functional proteins. Three representative examples

illustrate that the outlined procedures are robust, making it possible to solve by Nanobody-assisted X-ray crystallography

in a time span of 6–12 months.

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675

peptides or intrinsically unfolded parts of proteins. For linear

epitopes, conventional antibodies may be a better alternative.

Applications

The Nanobodies to be used as crystallization chaperones can also

be valuable for other applications in structural biology. For exam-

ple, domain-specific Nanobodies have been used in single-particle

electron microscopy (EM) as a marker to track these domains

in particle projections

34,35

. Because many Nanobodies can be

functionally expressed as intrabodies in eukaryotic cells, these

single-domain antibodies can also be used as biosensors to track

conformational properties of their targets inside a living cell

36–39

Ultimately, Nanobodies that constrain protein targets in unique

disease-linked conformations may facilitate the discovery of new

therapeutic molecules

Experimental design

General considerations. The workflow for generating, isolating

and characterizing the Nanobodies to be used as crystallization

chaperones (Fig. 1) is inherently dependent on the nature of the

antigen and on the purpose of the structural study. Several steps

in the Nanobody discovery process, including the preparation of

the immunogen, the selection strategy, the screening approach

and the functional and biophysical characterization, differ when

the target is a soluble protein, a membrane protein or a multi-

protein complex. To make this protocol broadly applicable for

the structural biology community, several modifications to the

standard protocol are referred to in the text.

Protein production and antigen quality control. Today, research-

ers are faced with a bewildering array of methods to produce and

purify recombinant proteins

. Although this is not the focus of

this paper, the supply of properly folded protein is crucial for the

generation of conformational Nanobodies. We discourage immu-

nization of animals with poorly characterized protein samples.

Our standard protocol typically requires 1 mg of the purified

protein: 700 µg for immunization and the remaining portion

for all Nanobody selection, identification and characterization

efforts. Ideally, a single batch of the purified protein is dispensed

into aliquots and stored under conditions that ensure the sta-

bility of the protein over time. Often, flash-freezing in liquid

nitrogen and storage at −80 °C is favored. We insist on confirm-

ing protein quality of one thawed aliquot before the protein is

administered as an immunogen. Biochemical or cellular assays

that quantitatively assess the functionality of the antigen (e.g.,

enzymatic and signaling activity, interaction with (radio)ligands

or binding of certified conventional antibodies against discon-

tinuous epitopes) can be used to test whether your protein is

properly folded. If your protein cannot be tested functionally,

we advise performing a rigorous biophysical characterization to

confirm its folded state (reviewed in ref. 42). Unless it has been

demonstrated not to have any effect on the functionality of the

protein, we strongly discourage multiple freeze-thaw cycles in

order to minimize protein denaturation. If samples cannot be

frozen, consider using freshly prepared protein throughout the

immunization and discovery effort.

Conformational locking of the antigens. Nanobodies have been

shown to stabilize proteins such as kinases and G protein–coupled

receptors (GPCRs) in unique biologically relevant conforma-

tions

33,43,44

. To identify such Nanobodies, we advise immunizing

animals with proteins constrained in the desired conformation

with cofactors, enzyme inhibitors

, orthosteric ligands

, allosteric

ligands or any molecule that conformationally traps the macro-

molecule in a particular state

. We recommend using ligands

that dissociate slowly to maximize the lifetime of the constrained

target conformation in the immunized animal. Purified, detergent-

solubilized membrane proteins may denature after immunization

owing to the dissociation of detergents. To maintain a native con-

formation after immunization, reconstitution of the protein into

a lipid environment, typically phospholipid vesicles

, or the use of

a very tight binding detergent

may be required. Reconstitution

of membrane proteins in lipid vesicles may also reduce ligand

dissociation. A ligand trapped in the vesicle can rebind the intra-

vesicular binding site of the protein, prolonging the extravesicular

exposure of the desired conformational state to the immune system.

Alternatively, particular mutations may trap the target in a

unique conformational state. Finally, chemical cross-linking

between protein domains or different proteins may stabilize

epitopes that are unique to the complex

Immunogen preparation, camelid immunization and repertoire

cloning. Antibodies with a homodimeric heavy-chain composition

devoid of light chains are only found in Tylopoda (camels, drom-

edaries and llamas) and sharks

. Our protocols can be applied to

llamas, camels, dromaderies and alpacas. All vaccination experi-

ments are executed according to EU animal welfare legislation and

after approval of the local ethics committee. It is known that stress

can cause immunosuppression. Animals should be manipulated

by authorized staff, preferably by an experienced veterinarian.

We allow animals to acclimatize to new housing conditions for

at least 1 week before immunization starts.

Protein production

Antigen quality control

LIama immunization

Functional antigen immobilization

Selection by phage display

Selection by yeast or bacterial display

Screening for antigen binders

Nanobody purification

Co crystallization

Structure refinement

Nanobody engineering for other biophysical applications

Biochemical and biophysical

characterization of binders

Repertoire cloning &

library construction

Reconstitution of antigen

in liposomes

Genetic and cell-based

vaccinations

(Bio)chemical cross-linking of

multiprotein antigens

Conformational locking of

the antigen

Step 1

Step 4

Step 7

Step 46

Step 62

Step 70

Figure 1

Workflow for the generation of conformational Nanobodies for

structural biology.

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In llamas, repetitive immunogen administrations generate a

robust immune response mediated by both conventional and

heavy chain–only antibodies. On the basis of 10 years of expe-

rience, GERBU LQ is a good immunostimulating adjuvant for

conformational Nanobody discovery, and it is well tolerated by

llamas. In general, we inject our animals subcutaneously with

cocktails of 1–5 antigens mixed with adjuvant. Alternatively,

different immunogens can be injected separately at different s.c.

locations. If small-molecule ligands are used for the conforma-

tional locking of an antigen, we always add these compounds in

excess to the antigen. In our hands, we have successfully reused

llamas for different Nanobody discovery projects by respecting a

grace period of at least 6 months.

A blood sample of 100 ml from an immunized llama contains suf-

ficient expressing B cells to clone a diverse set of the affinity-matured

Nanobodies with high specificity for their cognate antigen

Peripheral blood lymphocytes (PBLs) should be isolated without

delay from the noncoagulated blood for the purification of total

RNA and the synthesis of cDNA. From this cDNA, the Nanobody-

encoding open reading frames can be amplified by PCR and

cloned into an appropriate phage display vector.

Primer design. Over the years, several PCR strategies have been

developed to amplify Nanobody gene fragments from lymphocyte

cDNA. We prefer to use a two-step nested PCR approach. One

pair of primers (CALL001 and CALL002) has been designed for

the first PCR by using the cDNA of B lymphocytes as the tem-

plate

. The CALL002 primer anneals in a region of the second

constant heavy-chain domain (CH2) that is conserved among

all IgG isotypes of all camelids, whereas the CALL001 primer

anneals in a well-conserved region of the leader signal sequence

of all V elements of family III (by far the most abundant V fam-

ily in camelids). The primers VHH-Back and VHH-For are

used to amplify the Nanobody repertoire via a second nested

PCR (Supplementary Fig. 1a). From our experience, the prim-

ers described in PROCEDURE Steps 21 and 24 are adequate to

amplify Nanobody-encoding genes of family III from dromedary

(Camelus dromedaries), camel (Camelus bactrianus), llama (Lama

glama), and also alpaca (Vicugna pacos).

Van der Linden et al.

developed dedicated primers annealing

to the hinge of each heavy chain–only IgG isotype

of all camelid

species (Supplementary Fig. 1b). Maass et al.

designed prim-

ers dedicated for alpaca Nanobodies (Supplementary Fig. 1c).

Kastelic et al.

used primers that amplify mixtures of the VHH

(variable fragment of the heavy-chain antibody) and the VH

(variable fragment of the classical antibody) domains from llama

(Supplementary Fig. 1d).

Selection by phage display. Many excellent reviews on selection

methodologies to enrich for target-specific antibodies against

native epitopes have been published

. Phage display is certainly

the most robust technique, but yeast display

53,54

or bacterial

display

can also be used to select Nanobodies from immune

libraries. For conformational binders, it is essential to perform

the in vitro selection (panning) experiments under conditions

(buffer with appropriate detergent, pH, temperature and cofac-

tors) that produce the desired conformation of the protein during

phage incubation. To reduce the background of phage-expressing

nonspecific Nanobodies, the target protein should be highly pure

(>95%) and homogeneous in conformation.

Aspecific adsorption onto the solid surface of an ELISA plate is

still the most common way to immobilize targets for selection by

phage display, but this method can result in (partial) denaturation

of the protein

. Although the use of streptavidin-coated magnetic

beads is a valid alternative, we perform most panning experiments

in 96-well plates, which allows multiple parallel selection condi-

tions. Our preferred method is to capture biotinylated or tagged

target protein on a solid phase coated with NeutrAvidin or a tag-

specific antibody, respectively. Alternative strategies for presenting

target proteins during selection are summarized in Table 1. The

structural integrity and homogeneity of the presented target is

the most decisive factor for selecting Nanobodies with the desired

properties. If possible, we try different capturing or immobiliza-

tion methods, vary the antigen concentration, use different deter-

gents or ligands to keep the target in the desired conformation, try

different washing and incubation buffers or use different elution

methods in parallel pannings by using different selection wells on

the same 96-well plate. In many cases, magnetic beads can be used

as a valid alternative in order to perform selections in solution.

Depending on the magnitude of the heavy chain–only

antibody-mediated humoral response in the llama, typically

one or two rounds of panning are sufficient to enrich for target-

specific Nanobodies. Rather than selecting for target specificity

alone, we prefer implementing conditions early on that allow

the identification of Nanobodies with the desired functional or

biophysical properties: high affinity, stabilization of a unique

protein conformation, modulation of receptor function, binding

to a particular target domain or epitope, interference with

TABLE 1

Target presentation methods for the identification of

Nanobodies with demonstrated crystallization chaperone activity.

Target selection format Negative control

Biotinylated target protein

captured onto a

NeutrAvidin-coated well

Membrane protein that is

packed into a (biotinylated)

nanodisc and captured onto a

NeutrAvidin- or antibody-coated

well

(see Anticipated Results)

Irrelevant membrane

protein reconstituted in

(biotinylated) nanodiscs

and captured onto

a NeutrAvidin- or

antibody-coated well

Protein captured onto an

immobilized monoclonal antibody

that is specific for your protein

or a protein tag (His-tag,

Strep-tag, GST, etc.)

Well that is coated with

the monoclonal antibody

Solid-phase immobilized protein

by aspecific adsorption

6,70

Empty well

Solid-phase immobilized membrane

protein reconstituted in lipid

vesicles

(see Anticipated Results)

Well coated with lipid

vesicles harboring an

irrelevant membrane protein

Solid-phase immobilized membrane

protein that is packed into

virus-like particles

Well coated with an

irrelevant virus-like particle

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(orthosteric) ligand binding and stabilization of a protein com-

plex, among others. If no purified protein with demonstrated

functionality is available, Nanobody-panning methodologies that

use target-expressing cells (or derivatives) have been reported and

are implemented in our laboratory

Screening and functional/biophysical characterization of

conformational Nanobodies. After panning, we routinely

pick 96 individual clones from distinct selection outputs and

express the Nanobodies in the E. coli periplasm in 96-well plates.

In the past 10 years, we have rarely found Nanobodies that

bind linear epitopes. Therefore, we discourage the use of western

blotting or any other technique that tends to unfold the target.

In parallel to the assessment of antigen specificity, assays should

be implemented for the identification of Nanobodies with the

desired functional or biophysical properties. If the target is a

membrane protein, we recommend additionally assessing

Nanobody binding to target-overexpressing cells via fluorescence-

activated flow cytometry.

Our display vectors permit the inducible periplasmic expression

of Nanobodies as soluble C-terminally His

-tagged proteins in E. coli

strain WK6. Milligram quantities of >95% pure Nanobody can rou-

tinely be obtained by immobilized metal ion affinity chromatography

(IMAC) from the periplasmic extract of a 1-liter bacterial culture

As it is not known upfront which Nanobodies will behave as

the best crystallization chaperones, it is crucial to identify a large

panel of sequence-diverse Nanobodies (see Anticipated Results).

Typically, after a protein-based immunization and selection

discovery, between 3 and 30 Nanobody families are identified.

A Nanobody family is defined as a group of Nanobodies with

a high similarity in their CDR3 sequence (identical length and

>80% sequence identity). Nanobodies from the same family

derive from the same B-cell lineage and bind to the same epitope

on the target.

Nanobodies as crystallization chaperones. Electrophoretic

mobility shift assays on native gels provide a quick and easy

strategy for verifying whether purified Nanobodies form a

homogeneous complex with the target protein. Figure 2 shows how

mobility shift assays have been used to characterize Nanobodies

that bind an editosome protein of T. brucei

. Cocrystallization

experiments can be successful just by mixing the Nanobody and

target protein in a 1.2:1 molar ratio. Alternatively, the complex

can be further purified by size-exclusion chromatography after

co-incubation. Because Nanobodies are resistant to additives,

extremes of pH and temperature, and proteases

, they are

ideally suited for screening a broad range of crystallization

conditions with variations in pH, ionic strength, temperature,

protein concentration, salts, ligands or additives, and type of

precipitant. Conditions that can be screened are limited by the

stability of the target protein rather than by the stability of the

crystallization chaperones. More important is that Nanobodies

are extremely soluble proteins (≥40 mg ml

−1

), maximizing the

chance that they crystallize in complex with their cocrystallization

target rather than yielding Nanobody-only crystals. Because

they are efficiently produced in the periplasm of E. coli, SeMet-

labeled Nanobodies may ultimately be used for phasing via

single-anomalous dispersion technique without the need for

introducing SeMet into the target protein

. Alternatively, phase

information can be obtained from a molecular replacement

solution of the Nanobody in the complex.

Considerations about the laboratory facilities. We advise

dedicating two separate laboratories for Nanobody discovery.

One phage-free laboratory is used for Nanobody repertoire

amplification and cloning. We use dedicated reagents for library

construction. The other laboratory is used for all phage work,

including the amplification of phage and pannings. To reduce

phage contamination, we use filter tips and clean benches, equip-

ment and glassware with 1% (wt/vol) sodium hypochlorite

after each experiment. Use disposables whenever possible, and

discard them in 1% (wt/vol) sodium hypochlorite to inactivate

the remaining phage particles.

MATERIALS

REAGENTS

Llamas ! CAUTION All vaccination experiments should be executed in

accordance with the applicable animal welfare legislation, and they must be

approved by the local ethics committee.

Acetic acid, glacial (Merck, cat. no. 1.00058.2500) ! CAUTION Glacial acetic

acid is corrosive. Avoid inhalation and exposure to skin and eyes.

AEBSF protease inhibitor (Carl Roth, cat. no. 2931.3)

Agarose MP (Roche, cat. no. 11 388 991 001)

•

–

– –

+ +

Nanobody

Target protein

Target-Nb7 complex

Target-Nb8 complex

Target alone

Nb7

Target alone

Nb8

Figure 2

Native gel analysis of Nanobodies interacting with the

oligonucleotide-binding fold of the A1 protein of the editosome of the

sleeping sickness parasite Trypanosoma brucei

. Nanobodies Nb7 and Nb8

were generated with this protocol. The target protein was incubated with

Nb7 or Nb8 for 30 min at 4 °C in 20 mM Tris (pH 7.5), 1 mM DTT and

300 mM NaCl. Complex formation of target and Nanobody was analyzed on

a 4–15% (wt/vol) native gel at 110 V for 1 h and stained with Coomassie

Blue. The positions of the target protein alone (first lane), the target-Nb7

complex (third lane), the Nanobody Nb8 alone (fourth lane) and the

target-Nb8 complex (fifth lane) are indicated with symbols above the bands.

Owing to its high isoelectric point, Nb7 is too positively charged to run into

the native gel (second lane).

AP-conjugate substrate: 4-nitrophenyl phosphate disodium salt hexahydrate

(DNPP, Sigma-Aldrich, cat. no. 71768)

CaCl

·2H

O (Merck, cat. no. 2382)

Chloroform (Merck, cat. no. 1.02445.0250)

Citric acid·H

O (Sigma-Aldrich, cat. no. C1909)

E. coli strains  CRITICAL Use an amber codon suppression (supE)

strain such as TG1 (electrocompetent cells ≥4 × 10

c.f.u. µg

−1

;

Lucigen, cat. no. 60502-1) for cloning and preparation of phage libraries,

•

A general protocol for the generation of Nanobodies for structural biology

Figures

Citations

Structural insights into µ-opioid receptor activation

The P2X7 Receptor Channel: Recent Developments and the Use of P2X7 Antagonists in Models of Disease

Nanobodies as therapeutics: big opportunities for small antibodies

Structure of the Nanobody-Stabilized Active State of the Kappa Opioid Receptor

Distinct conformations of GPCR–β-arrestin complexes mediate desensitization, signaling, and endocytosis

References

Naturally occurring antibodies devoid of light chains

Crystal structure of the β2 adrenergic receptor-Gs protein complex.

The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on

Structure of a nanobody-stabilized active state of the β2 adrenoceptor

Nanobodies: Natural Single-Domain Antibodies

Related Papers (5)

Nanobodies: Natural Single-Domain Antibodies

Naturally occurring antibodies devoid of light chains

Structure of a nanobody-stabilized active state of the β2 adrenoceptor

Features and development of Coot.

Phaser crystallographic software

Frequently Asked Questions (15)

Q1. What are the contributions in this paper?

Q2. What is the way to stabilize epitopes?

Q3. Why do in vivo-matured antibodies have the ability to bind antigen?

Q4. Why are SeMetlabeled Nanobodies used in E. coli?

Q5. What is the significance of the periplasmic expression of Nanobodies?

Q6. What is the important factor for selecting Nanobodies?

Q7. What is the role of nanobodies in the crystallization of proteins?

Q8. What is the way to test the ahomogeneous complex of Nanobodies?

Q9. how many rounds of panning are sufficient to enrich for targetspecific Nanobodies?

Q10. How many bp of cDNA should be amplified?

Q11. How do you remove the phage particles from the wells?

Q12. How can nanobodies be incorporated into the crystallization pipeline?

Q13. How many dN6 random primers are stored in a sigma-a?

Q14. What is the robust technique for phage display?

Q15. What is the workflow for generating, isolating and characterizing the nanobodies?