From Llama to Nanobody: A Streamlined Workflow for the Generation of Functionalised VHHs
从美洲驼到纳米体：生成功能化VHHs的简化工作流程
发布: 2024年03月20日第14卷第6期 DOI: 10.21769/BioProtoc.4962 浏览次数: 7040
评审: Luis Alberto Sánchez VargasJaveena HussainDogan Can KirmanThirupugal Govindarajan
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Abstract
Nanobodies are recombinant antigen-specific single domain antibodies (VHHs) derived from the heavy chain–only subset of camelid immunoglobulins. Their small molecular size, facile expression, high affinity, and stability have combined to make them unique targeting reagents with numerous applications in the biomedical sciences. From our work in producing nanobodies to over sixty different proteins, we present a standardised workflow for nanobody discovery from llama immunisation, library building, panning, and small-scale expression for prioritisation of binding clones. In addition, we introduce our suites of mammalian and bacterial vectors, which can be used to functionalise selected nanobodies for various applications such as in imaging and purification.
Key features
• Standardise the process of building nanobody libraries and finding nanobody binders so that it can be repeated in any lab with reasonable equipment.
• Introduce two suites of vectors to functionalise nanobodies for production in either bacterial or mammalian cells.
Graphical overview
Nanobody (纳米体)
Phage display (噬菌体展示)
Nanobody labelling (纳米体标记)
Background
The fact that camelids produce a unique heavy-chain antibody was discovered serendipitously some thirty years ago by researchers at the Vrije Universiteit Brussel [1]. Pioneering work from the Belgian group showed that the variable heavy chain domain of these antibodies, termed VHH, could be produced as a single domain binding protein, referred to as a nanobody. Subsequently, nanobodies have been generated to a wide variety of antigens for applications in cell and structural biology, including as crystallization chaperones for high-value membrane and unstructured proteins and as probes for super-resolution microscopy [2]. Typically, nanobodies are generated by screening phage display libraries of VHH domains cloned from the peripheral blood cells of immunised llamas and alpacas with the target immunogen [3]. Since 2019, our group have generated nanobodies to 75 different antigens. The antigens varied from complexes, membranes, and soluble proteins. Some of the nanobodies that we have identified have been applied as structural chaperones [4], diagnostics [5], and anti-viral therapeutics for SARS-CoV-2 [6] and for in vitro and in vivo cell biology [7]. Although several protocols for producing nanobodies have been published previously [8], we have identified areas for streamlining the process, e.g., incorporating ligation-independent cloning to facilitate the construction of VHH domain libraries and small-scale expression screening to identify high-producing clones.
Furthermore, by using generic VHH cloning primers, we have designed a suite of expression vectors that enable the functionalisation of any cloned VHH with a variety of carboxy terminal tags, e.g., Flag, Avi-tag®, or SNAP-tag® for subsequent site-specific labelling. Overall, our aim is to make this technology readily accessible to any research group with an appropriately equipped laboratory.
Materials and reagents
Biological materials
Post-immune heparinised llama blood
Pre- and post-immune llama sera
Electrocompetent TG1 (Agilent, catalog number: 200123)
CM13K trypsin-sensitive helper phages (Antibody Design Laboratories, catalog number: PH050L)
StellarTM competent cells (Takara Bio, catalog number: 636766)
Escherichia coli (Migula) Castellani and Chalmers, strain WK6 (ATCC, catalog number: 47078)
BL21(DE3)-R3-pRARE2-BirA E. coli cells for in vivo biotinylation, SGC (Structural Genomics Consortium)
HeLa cells (ATCC, catalog number: CCL-2)
Reagents
Gerbu adjuvant F (Biotechnik GmbH, catalog number: 3030). Store at 4 °C
NeutrAvidinTM biotin-binding protein (Invitrogen, catalog number: A2666). Store at -20 °C
10× PBS (Fisher BioReagents, catalog number: BP399-20), for general use. Store at room temperature (RT)
Skim milk powder (Oxoid, catalog number: LP0031). Store at RT
Tween-20 (Sigma-Aldrich, catalog number: P1379). Store at RT
BSA (Sigma-Aldrich, catalog number: A2153). Store at 4 °C
Goat anti-llama IgG (H+L) HRP (Invitrogen, catalog number: A16060). Store at -20 °C in 5 µL aliquots
KPL ABTS peroxidase solution A (SeraCare, catalog number: 5120-0034). Store at 4 °C
KPL peroxidase substrate solution B (SeraCare, catalog number: 5120-0037). Store at 4 °C
ChemgeneTM (Chemgene&Trade, catalog number: XTM308). Store at RT
Caution: This chemical is corrosive.
Ethanol (EtOH) (Fisher BioReagents, catalog number: E/0650DF/17). Store at RT in a flammable liquid storage cupboard
Caution: This chemical is flammable.
Virkon® (Rely+onTM, catalog number: 12358667). Store at RT
Caution: This chemical is corrosive.
PBS, pH 7.4 for polymorphonuclear cell (PMNC) isolation (Gibco, catalog number: 10010015). Store at 4 °C
Histopaque®-1077 (Sigma-Aldrich, catalog number: 10771). Store at 4 °C
RNaseZapTM RNase decontamination solution (Invitrogen, catalog number: AM9782). Store at RT
Trypan blue solution, 0.4% (Sigma-Aldrich, catalog number: T8154-20ML). Store at RT
Caution: This chemical is potentially carcinogenic.
TRIzolTM reagent (Invitrogen, catalog number:15596026). Store at 4 °C
Caution: This chemical is corrosive and toxic.
Chloroform (Fluorchem, catalog number: D007F). Store at RT in a flammable liquid storage cupboard
Caution: This chemical is flammable.
2-propanol (Sigma-Aldrich, catalog number: I9516). Store at RT in a flammable liquid storage cupboard
Caution: This chemical is flammable.
Nuclease-free water (not DEPC treated) (Invitrogen, catalog number: AM9932). Store at RT
SuperScriptTM IV one-step RT-PCR system (Invitrogen, catalog number: 12594025). Store at -20 °C
CALL_001 primer 5′ GTCCTGGCTGCTCTTCTACAAGG 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
CALL_002 primer 5′ GGTACGTGCTGTTGAACTGTTCC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
6× DNA gel loading buffer (New England Biolabs, catalog number: B7024S). Store at 4 °C
10× TBE buffer (Thermo Scientific, catalog number: B52). Store at RT
Agarose (Fisher BioReagents, catalog number: BP1356-500). Store at RT
1,000× SYBRTM safe DNA gel stain (Invitrogen, catalog number: S33102). Store at RT
GeneRuler 1 kb DNA ladder (Thermo Scientific, catalog number: SM0311). Store at 4 °C
Nucleospin Gel and PCR clean-up kit (Macherey-Nagel, catalog number: 740609.50). Store at RT
Purelink PCR purification kit (Invitrogen, catalog number: K310002). Store at RT
HyperLadderTM 1 kb DNA ladder (Meridian Bioscience®, Bioline, catalog number: BIO-33053). Store at 4 °C
Phusion flash high fidelity PCR master mix (Invitrogen, catalog number: F548L). Store at -20 °C
VHHFor2 primer 5′ TACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCT GGRGGA 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
VHHRev primer 5′ GTGATGGTGTTGGCCTCCTGAGGAGACGGTGACCTGG 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
pADL23c vector (Antibody Design Laboratories, catalog number: PD0111). Store at -20 °C
SfiI (New England Biolabs, catalog number: R0123S). Store at -20 °C
10× rCutSmartTM buffer (New England Biolabs, catalog number: B6004S). Store at 4 °C
ClonExpress II one step cloning kit, which contains 5× CEII buffer and Exnase II (Vazymbiotech, catalog number: C112-02). Store at -20 °C
Recovery medium (Sigma-Aldrich, catalog number: S1797). Store at 4 °C
LB medium mix (Formedium, catalog number: LBL0103). Store at RT
Bacto tryptone (Melford, catalog number: T60065-2000.0). Store at RT
Yeast extract (Melford, catalog number: Y20020-1000.0). Store at RT
Bacto agar (Formedium, catalog number: AGR10). Store at RT
Ampicillin (Formedium, catalog number: AMP100). Store at 4 °C
Caution: Ampicillin is a sensitiser.
25 mM dNTP (Thermo Scientific, catalog number: R1122). Store at -20 °C in 30 µL aliquots
Taq polymerase with standard Taq reaction buffer (New England Biolabs, catalog number: M0273X). Store at -20 °C
PhD_seq_Fwd primer 5′ GCTTCCGGCTCGTATGTTG 3′. Store at -20 °C
PhD_seq_Rev primer 5′ GTCGTCTTTCCAGACGTTAG 3′. Store at -20 °C
Glycerol (Sigma-Aldrich, catalog number: G5516-1L). Store at RT
PEG 6000 (Sigma-Aldrich, catalog number: 81260). Store at RT
NaCl (Sigma-Aldrich, catalog number: S9888). Store at RT
Kanamycin (Sigma-Aldrich, catalog number: K1377). Store at 4 °C
Caution: Kanamycin is a sensitiser and may damage fertility.
StartingBlockTM (PBS) blocking buffer (Invitrogen, catalog number: 37538). Store at 4 °C
DynabeadsTM M-280 streptavidin (Invitrogen, catalog number: 11205D). Store at 4 °C
Tris base (Melford, catalog number: T60040-100.0). Store at RT
CaCl2·2H2O (Sigma-Aldrich, catalog number: C3306). Store at RT
Trypsin (Sigma-Aldrich, catalog number: T1426). Store at -20 °C
Anti-M13 HRP (Sino Biological, catalog number: 11973-MM05T-H). Store at -20 °C in 5 µL aliquots
Highprep PCR magnetic beads (Magbio, catalog number: AC60050). Store at 4 °C
Glucose (Sigma-Aldrich, catalog number: G8270). Store at RT
MgCl2·6H2O (Sigma-Aldrich, catalog number: M2670). Store at RT
IPTG (NeoBiotech, catalog number: NB-45-00030). Store at -20 °C
Polymyxin B sulfate (Gibco, catalog number: 21850029). Store at RT
Caution: Polymyxin B sulfate is toxic.
2× Laemmli buffer (Sigma-Aldrich, catalog number: S3401). Store at 4 °C
20× NuPAGETM MES SDS running buffer (Invitrogen, catalog number: NP000202). Store at RT
Mark12TM unstained standard (Invitrogen, catalog number: LC5677). Store at 4 °C
InstantBlue® Coomassie protein stain (Abcam, catalog number: ab119211). Store at 4 °C
Ni-NTA spin columns (QIAGEN, catalog number: 31014). Store at 4 °C
Imidazole (Sigma-Aldrich, catalog number: I202-500g). Store at RT
Caution: Imidazole is toxic, corrosive, and an irritant and may damage fertility.
ZebaTM spin desalting columns, 7K MWCO, 0.5 mL (Thermo Scientific, catalog number: 89882). Store at 4 °C
Rabbit anti-camelid VHH HRP (GenScript, catalog number: A01861-200). Store at -20 °C in 5 µL aliquots
pOPINVHH_his vector (Addgene, catalog number: 210405). Store at -20 °C
pOPINVHH_cys_his vector (Addgene, catalog number: 210403). Store at -20 °C
pOPINVHH_BAP_his vector (Addgene, catalog number: 210402). Store at -20 °C
pOPINVHH_sort_his vector (Addgene, catalog number: 210406). Store at -20 °C
pOPINVHH_flag_his vector (Addgene, catalog number: 210404). Store at -20 °C
pOPINVHH_myc_his vector (Addgene, catalog number: 210526). Store at -20 °C
pOPINVHH_snap_his vector (Addgene, catalog number: 210407). Store at -20 °C
pOPINE-3C-eGFP vector (Addgene, catalog number: 41125). Store at -20 °C
pOPINE-3C-eYFP vector (Addgene, catalog number: 214028). Store at -20 °C
pOPINE-3C-mCherry vector (Addgene, catalog number: 214060). Store at -20 °C
Common Fwd primer 5′ GCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGGAG 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
His FLAG Rev primer 5′ GTGATGGTGGCCTGAGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
Cys Rev primer 5′ ATGGTGACAGCCTGAGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
BAP Rev primer 5′ ATCATTCAAGCCTGAGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C.
Sort Rev primer 5′ CGGCAGGCCGCCTGAGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
Myc Rev primer 5′ GTGATGGTGGTGGCCTGAGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
SNAP Rev primer 5′ GTCCTTGTCGCCTGAGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
pOPINE common Fwd primer 5′ AGGAGATATACCATGCAGGTGCAGCTGGTGGAG 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
pOPINE common Rev primer 5′ CAGAACTTCCAGTTTAGGGGAGACGGTGACCTGGGTC 3′. Ordered primer from IDT. Store at RT prior to reconstitution in water, then store at -20 °C
Bsu36I (New England Biolabs, catalog number: R0524S). Store at -20 °C
NcoI (New England Biolabs, catalog number: R01093S). Store at -20 °C
X-gal ready to use (Thermo Scientific, catalog number: R0941). Store at 4 °C
QIAprep spin miniprep kit (QIAGEN, catalog number: 27104). Store at RT
DNA sequence of anti-GFP nanobody [9] after codon optimisation for expression in E. coli. CAG GTC CAA TTA GTG GAG TCC GGT GGG GCA CTT GTC CAG CCT GGA GGT TCA CTT CGC TTG TCT TGC GCA GCG TCT GGA TTC CCG GTG AAC CGC TAT AGT ATG CGT TGG TAC CGT CAA GCT CCG GGG AAA GAA CGT GAA TGG GTA GCA GGG ATG TCT TCC GCC GGT GAC CGC TCT TCA TAC GAG GAC TCG GTC AAG GGG CGC TTC ACA ATC TCT CGT GAT GAT GCC CGT AAC ACC GTT TAC TTG CAA ATG AAC AGC CTG AAA CCG GAA GAC ACT GCG GTG TAT TAC TGC AAT GTT AAT GTA GGG TTT GAA TAC TGG GGT CAA GGT ACA CAA GTT ACA GTT TCG TCA. Ordered gBlock from IDT. Store at RT prior to reconstitution in TE buffer, incubate at 50 °C for 20 min, then store at -20 °C
Chloramphenicol (Sigma-Aldrich, catalog number: C1919). Store at 4 °C
Caution: Chloramphenicol is toxic, can cause eye damage, and is a carcinogen.
Spectinomycin dihydrochloride pentahydrate (Sigma-Aldrich, catalog number: S4014). Store at 4 °C
Caution: Spectinomycin is an irritant.
Carbenicillin disodium salt (Sigma-Aldrich, catalog number: C3416). Store at 4 °C
Caution: Carbenicillin is a sensitiser.
Biotin (Sigma-Aldrich, catalog number: B4639). Store at 4 °C
Streptavidin, Alexa FluorTM 488 conjugate (Invitrogen, catalog number: S11223). Store at -20 °C
BenchMarkTM fluorescent protein standard (Invitrogen, catalog number: LC5928). Store at -20 °C
DNA sequence of anti-vimentin (VB3) nanobody [10] after codon optimisation for expression in E. coli: CAG GTC CAA CTT GTA GAG TCA GGA GGT GGA AGC GTG CAA GCT GGG GAC TCT CTG CGC CTG TCT TGT GCT TCG AGC GGA AAT ACC TTC TCG ATC AAA GTC ATG GGA TGG TAC CGC CAG GCA CCT GGA AAG CAA CGT GAA TTA GTC GCG GTT TCA ACC AAT AGC GGG GCC TCT GTT AAT TAT GCC AAC TCT GTG AAG GGA CGC TTT ACC ATT TCT ATT GAT TCA GTA AAA AAA ACA ACC TAC TTA CAG ATG AAT TCC TTG AAG CCA GAA GAT ACA GCC GTC TAC TTT TGC AAT GCA TAT GAT GGG CGT TAT GAG GAC TAT TAC GGT CAG GGG ACC CAA GTG ACA GTA TCA TCA. Ordered gBlock from IDT. Store at RT prior to reconstitution in TE buffer, incubate at 50 °C for 20 min, then store at -20 °C
TCEP, hydrochloride (Merck, Millipore®, catalog number: 580567-5GM). Store at RT
Alexa FluorTM 647 C2-maleimide (Invitrogen, catalog number: A20347). Store at -20 °C
ZebaTM dye and biotin removal columns (Thermo Scientific, catalog number: A44296). Store at 4 °C
PageRulerTM prestained protein ladder (Thermo Scientific, catalog number: 26616). Store at 4 °C
DMEM, high glucose, HEPES, no phenol red (Gibco, catalog number: 21063-029). Store at 4 °C
Fetal bovine serum (FBS) (Biowest, catalog number: S00NB1001Y). Store at -20 °C
100× GlutaMAXTM (Gibco, catalog number: 35050-038). Store at -20 °C
100× penicillin-streptomycin (Gibco, catalog number: 15140-122). Store at -20 °C
4% paraformaldehyde in PBS (Thermo Scientific chemicals, catalog number: J61899.AK). Store at 4 °C
Caution: Paraformaldehyde is an eye irritant, skin sensitiser, and carcinogen.
TritonTM X-100 (Sigma-Aldrich, catalog number: T8787-250mL). Store at RT
Fluoroshield with DAPI (Abcam, catalog number: ab104139). Store at RT
Nail varnish. Store at RT
Type F immersion liquid (Leica, catalog number: 11513859). Store at RT
Fugene transfection reagent (Promega, catalog number: E2311). Store at 4 °C
Solutions
0.5 mg/mL neutrAvidin biotin-binding protein (see Recipes)
10 µg/mL neutrAvidin for coating ELISA plate (see Recipes)
1× PBS (see Recipes)
Blocking solution (see Recipes)
Washing buffer (PBST) (see Recipes)
0.1% (w/v) BSA-PBS (see Recipes)
5% (v/v) Chemgene (see Recipes)
70% (v/v) ethanol (EtOH) (see Recipes)
2% (w/v) Virkon (see Recipes)
75% (v/v) EtOH (see Recipes)
1× TBE buffer (see Recipes)
0.7% (w/v) agarose gel containing 1× SYBRTM Safe DNA gel stain (see Recipes)
1% (w/v) agarose gel containing 1× SYBRTM Safe DNA gel stain (see Recipes)
2× YT medium (see Recipes)
LB medium (see Recipes)
100 mg/mL ampicillin (see Recipes)
1% (w/v) agar LB plates containing 100 µg/mL ampicillin (see Recipes)
2% (w/v) agar LB plates containing 100 µg/mL ampicillin (see Recipes)
50% glycerol (see Recipes)
2× YT medium containing 25% glycerol (see Recipes)
1% (w/v) agar LB plates without antibiotic (see Recipes)
2× YT containing 100 µg/mL ampicillin (see Recipes)
50 mg/mL kanamycin (see Recipes)
2× YT containing 25 µg/mL kanamycin (see Recipes)
PEG/NaCl precipitation solution (see Recipes)
2× YT containing 100 µg/mL ampicillin and 25 µg/mL kanamycin (see Recipes)
TBSC (see Recipes)
1 mg/mL trypsin (see Recipes)
250 µg/mL trypsin (see Recipes)
Terrific broth (see Recipes)
20% (w/v) glucose (see Recipes)
1 M MgCl2·6H2O (see Recipes)
Terrific broth containing 100 µg/mL ampicillin, 0.1% glucose, 2 mM MgCl2·6H2O (see Recipes)
1 M IPTG (see Recipes)
1 mg/mL polymyxin B sulfate in PBS (see Recipes)
1× NuPAGETM MES SDS running buffer (see Recipes)
Equilibration buffer (see Recipes)
Elution buffer (see Recipes)
Octet dilution buffer (see Recipes)
1% (w/v) agar LB plate containing 100 µg/mL ampicillin, 2 mM IPTG, 40 µg/mL X-gal (see Recipes)
34 µg mL chloramphenicol (see Recipes)
50 µg/mL spectinomycin (see Recipes)
50 µg/mL carbenicillin (see Recipes)
1% (w/v) agar LB plate containing 34 µg/mL chloramphenicol, 50 µg/mL spectinomycin, 50 µg/mL carbenicillin (see Recipes)
0.2 M Biotin (see Recipes)
Terrific broth containing 50 µg/mL spectinomycin and 50 µg/mL carbenicillin (see Recipes)
1 mg/mL Streptavidin Alexa FluorTM 488 conjugate (see Recipes)
50 µg/mL Streptavidin Alexa FluorTM 488 conjugate (see Recipes)
500 mM TCEP, pH 8.0 (see Recipes)
1 mM TCEP, pH 8.0 (see Recipes)
10 mg/mL Alexa FluorTM 647 C2-maleimide (see Recipes)
DMEM medium without phenol red, 10% (v/v) FBS, 1× GlutaMAXTM, 1× Penicillin-streptomycin (see Recipes)
Confocal blocking buffer (see Recipes)
Confocal dilution buffer (see Recipes)
Recipes
0.5 mg/mL neutrAvidin biotin-binding protein
Prepare 200 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume neutrAvidinTM 0.5 mg 5 mg ddH2O n/a 10 mL Total n/a 10 mL 10 µg/mL neutrAvidin for coating ELISA plate
Prepare just before use.
Reagent Final concentration Quantity or Volume neutrAvidinTM (Recipe 1) 10 µg/mL 200 µL PBS (1×) 1× 9.8 mL Total n/a 10 mL 1× PBS
Prepare both sterile (by autoclaving) and non-sterile PBS. Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume PBS (10×) 1× 100 mL ddH2O n/a 900 mL Total n/a 1,000 mL Blocking solution
Prepare on day of use.
Reagent Final concentration Quantity or Volume Milk 2% (w/v) 2 g PBS (Recipe 3) (or buffer that is compatible with the protein) n/a 100 mL Total n/a 100 mL Washing buffer (PBST)
Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume PBS (Recipe 3) 1× 1 000 mL Tween-20 0.05% (v/v) 500 µL Total n/a 1,000 mL 0.1% (w/v) BSA-PBS
Prepare on day of use.
Reagent Final concentration Quantity or Volume BSA 0.1 % (w/v) 50 mg PBS (Recipe 3) 1× 50 mL Total n/a 50 mL 5% (v/v) Chemgene
Store at RT.
Reagent Final concentration Quantity or Volume Chemgene (100%) 5% (v/v) 50 mL H2O n/a 950 mL Total n/a 1,000 mL 70% (v/v) EtOH
Store at RT.
Reagent Final concentration Quantity or Volume Ethanol (absolute) 70% (v/v) 70 mL H2O n/a 30 mL Total n/a 100 mL 2% (w/v) Virkon
Prepare weekly and store at RT.
Reagent Final concentration Quantity or Volume Virkon powder 2% (w/v) 100 g H2O n/a 5,000 mL Total n/a 5,000 mL 75% (v/v) EtOH
Store at RT.
Reagent Final concentration Quantity or Volume Ethanol (absolute) 75% (v/v) 75 mL H2O n/a 25 mL Total n/a 100 mL 1× TBE buffer
Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume TBE (10×) 1× 100 mL H2O n/a 900 mL Total n/a 1,000 mL 0.7% (w/v) agarose gel containing 1× SYBRTM Safe DNA gel stain
Heat in a microwave until all the agarose has dissolved and allow to cool to 55 °C before the addition of the gel stain. Pour into the DNA casting apparatus and allow to solidify. Prepare on day of use.
Reagent Final concentration Quantity or Volume Agarose 0.7 % (w/v) 0.7 g TBE (Recipe 11) 1× 100 mL Total n/a 100 mL SYBRTM Safe DNA gel stain (1,000×) 1× 10 µL 1% (w/v) agarose gel containing 1× SYBRTM Safe DNA gel stain
Heat in a microwave until all the agarose has dissolved and allow to cool to 55 °C before the addition of the gel stain. Pour into the DNA casting apparatus and allow to solidify. Prepare on day of use.
Reagent Final concentration Quantity or Volume Agarose 1% (w/v) 1 g TBE (Recipe 11) 1× 100 mL Total n/a 100 mL SYBRTM Safe DNA gel stain (1,000×) 1× 10 µL 2× YT medium
Autoclave and store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume Bacto tryptone 16 g/L 4 g NaCl 5 g/L 1.25 g Yeast extract 10 g/L 2.5 g ddH2O n/a 250 mL Total n/a 250 mL LB medium
Autoclave and store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume LB medium mix 25 g/L 6.25 g ddH2O n/a 250 mL Total n/a 250 mL 100 mg/mL ampicillin
Filter sterilise using a 0.22 µm filter. Prepare 500 µL aliquots and store at -20 °C. Stable for at least one year.
Caution: Ampicillin is a sensitiser.
Reagent Final concentration Quantity or Volume Ampicillin 100 mg/mL 2 g ddH2O n/a 20 mL Total n/a 20 mL 1% (w/v) agar LB plates containing 100 µg/mL ampicillin
Autoclave LB medium and bacto agar mix and allow to cool to 55 °C before the addition of antibiotic. Invert to mix and pour into 8.5 cm Petri dishes in a safety cabinet. Allow to cool at RT. Prepare on day of use.
Reagent Final concentration Quantity or Volume LB medium mix 25 g/L 6.25 g Bacto agar 1% (w/v) 2.5 g ddH2O n/a 250 mL Total n/a 250 mL Ampicillin (Recipe 16) 100 µg/mL 250 µL 2% (w/v) agar LB plates with 100 µg/mL ampicillin
Autoclave LB medium and bacto agar mix and allow to cool to 55 °C before the addition of antibiotic. Invert to mix and pour into 8.5 cm Petri dishes in a safety cabinet. Allow to cool at RT. Prepare on day of use.
Reagent Final concentration Quantity or Volume LB medium mix 25 g/L 6.25 g Bacto agar 2% (w/v) 5 g ddH2O n/a 250 mL Total n/a 250 mL Ampicillin (Recipe 16) 100 µg/mL 250 µL 50% (v/v) glycerol
Autoclave and store at RT. Stable for at least one year.
Reagent Final concentration Quantity or Volume Glycerol 50% (v/v) 100 mL ddH2O n/a 100 mL Total n/a 200 mL 2× YT medium containing 25% (v/v) glycerol
Prepare just before use in a safety cabinet.
Reagent Final concentration Quantity or Volume 2× YT medium n/a 20 mL Glycerol (Recipe 19) 25% (w/v) 20 mL Total n/a 40 mL 1% (w/v) agar LB plates without antibiotic
Autoclave and allow to cool to 55 °C. Invert to mix and pour into 8.5 cm Petri dishes in a safety cabinet. Allow to cool and set at RT. Store at 4 °C for two weeks.
Reagent Final concentration Quantity or Volume LB medium mix 25 g/L 6.25 g Bacto agar 1% (w/v) 2.5 g ddH2O n/a 250 mL Total n/a 250 mL 2× YT containing 100 µg/mL ampicillin
Prepare just before use in a safety cabinet.
Reagent Final concentration Quantity or Volume 2× YT n/a 100 mL Ampicillin (Recipe 16) 100 µg/mL 100 µL Total n/a 100 mL 50 mg/mL kanamycin
Filter sterilise using a 0.22 µm filter. Prepare 100 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume Kanamycin 50 mg/mL 0.5 g ddH2O n/a 10 mL Total n/a 10 mL 2× YT containing 25 µg/mL kanamycin
Prepare just before use in a safety cabinet.
Reagent Final concentration Quantity or Volume 2× YT n/a 100 mL Kanamycin (Recipe 23) 25 µg/mL 50 µL Total n/a 100 mL PEG/NaCl precipitation solution
Autoclave and store at RT. Stable for at least one year.
Reagent Final concentration Quantity or Volume PEG 6000 20% (w/v) 200 g NaCl 2.5 M 146.1 g ddH2O n/a Make up to 1,000 mL Total n/a 1,000 mL 2× YT containing 100 µg/mL ampicillin and 25 µg/mL kanamycin
Prepare just before use in a safety cabinet.
Reagent Final concentration Quantity or Volume 2× YT n/a 100 mL Ampicillin (Recipe 16) 100 µg/mL 100 µL Kanamycin (Recipe 23) 25 µg/mL 50 µL Total n/a 100 mL TBSC
Autoclave and store at RT. Stable for at least one year.
Reagent Final concentration Quantity or Volume Tris base 10 mM 0.788 g NaCl 137 mM 4 g CaCl2·2H2O 1 mM 73.5 mg ddH2O n/a 500 mL Total n/a 500 mL 1 mg/mL trypsin
Prepare 130 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume Trypsin 1 mg/mL 50 mg ddH2O n/a 50 mL Total n/a 50 mL 250 µg/mL trypsin
Prepare just before use.
Reagent Final concentration Quantity or Volume Trypsin (Recipe 28) 250 µg/mL 125 µL TBSC (Recipe 27) n/a 375 µL Total n/a 500 µL Terrific broth
Autoclave and store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume Bacto tryptone 12 g/L 3 g Yeast extract 24 g/L 6 g Glycerol 0.4% (v/v) 1 mL KH2PO4 0.17 M 0.5775 g K2HPO4 0.72 M 3.135 g ddH2O n/a 250 mL Total n/a 250 mL 20% (w/v) glucose
Filter sterilise using a 0.22 µm filter. Prepare 500 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume Glucose 20% (w/v) 20 g ddH2O n/a Make up to 100 mL Total n/a 100 mL 1 M MgCl2·6H2O
Filter sterilise using a 0.22 µm filter. Prepare 500 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume MgCl2·6H2O 1 M 4.066 g ddH2O n/a 20 mL Total n/a 20 mL Terrific broth containing 100 µg/mL ampicillin, 0.1% glucose, 2 mM MgCl2·6H2O
Prepare just before use in a safety cabinet.
Reagent Final concentration Quantity or Volume Terrific broth (Recipe 30) n/a 99.2 mL Ampicillin (Recipe 16) 100 µg/mL 100 µL Glucose (Recipe 31) 0.1% (v/v) 500 µL MgCl2·6H2O (Recipe 32) 2 mM 200 µL Total n/a 100 mL 1 M IPTG
Filter sterilise using a 0.22 µm filter. Prepare 500 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume IPTG 1 M 4.766 g ddH2O n/a 20 mL Total n/a 20 mL 1 mg/mL polymyxin-B sulfate in PBS
Prepare 1 mL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume Polymyxin-B sulfate 1 mg/mL 2 mg PBS (Recipe 3) 1× 2 mL Total n/a 2 mL 1× NuPAGETM MES SDS running buffer
Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume MES (20×) 1× 100 mL H2O n/a 1,900 mL Total n/a 2,000 mL Equilibration buffer
Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume Imidazole 30 mM 0.51 g PBS (Recipe 3) 1× 250 mL Adjust pH to 7.4 Total n/a 250 mL Elution buffer
Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume Imidazole 300 mM 2.04 g PBS (Recipe 3) 1× 100 mL Adjust pH to 7.4 Total n/a 100 mL Octet dilution buffer
Store at RT. Stable for at least one month.
Reagent Final concentration Quantity or Volume BSA 0.1% (w/v) 50 mg PBS (Recipe 3) (or buffer that is compatible with the protein) 1× 50 mL Total n/a 50 mL 1% (w/v) agar LB plate containing 100 µg/mL ampicillin, 2 mM IPTG, 40 µg/mL X-gal
Autoclave and allow to cool to 55 °C before the addition of additives. Invert to mix and pour into 8.5 cm Petri dishes in a safety cabinet. Allow to cool and set at RT. Store at 4 °C for two weeks.
Reagent Final concentration Quantity or Volume LB medium mix 25 g/L 6.25 g Bacto agar 1% (w/v) 2.5 g ddH2O n/a Make up to 250 mL Total n/a 250 mL Ampicillin (Recipe 16) 100 µg/mL 250 µL IPTG (Recipe 34) 2 mM 500 µL X-gal (20 mg/mL) 40 µg/mL 500 µL 34 mg/mL chloramphenicol
Filter sterilise using a 0.22 µm filter. Prepare 100 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume Chloramphenicol 34 mg/mL 34 mg EtOH 100% 1 mL Total n/a 1 mL 50 mg/mL Spectinomycin
Filter sterilise using a 0.22 µm filter. Prepare 100 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume Spectinomycin dihydrochloride pentahydrate 50 mg/mL 50 mg ddH2O n/a 1 mL Total n/a 1 mL 50 mg/mL Carbenicillin
Filter sterilise using a 0.22 µm filter. Prepare 100 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume Carbenicillin disodium salt 50 mg/mL 50 mg ddH2O n/a 1 mL Total n/a 1 mL 1% (w/v) agar LB plate containing 34 µg/mL chloramphenicol, 50 µg/mL spectinomycin, 50 µg/mL carbenicillin
Autoclave LB medium and bacto agar mix and allow to cool to 55 °C before the addition of additives. Invert to mix and pour into 8.5 cm Petri dishes in a safety cabinet. Allow to cool and set at RT. Store at 4 °C for two weeks.
Reagent Final concentration Quantity or Volume LB medium mix 25 g/L 2.5 g Bacto agar 1% (w/v) 1 g ddH2O n/a Make up to 100 mL Total n/a 100 mL Chloramphenicol (Recipe 41) 34 µg/mL 100 µL Spectinomycin (Recipe 42) 50 µg/mL 100 µL Carbenicillin (Recipe 43) 50 µg/mL 100 µL 0.2 M biotin
Filter sterilise using a 0.22 µm filter. Prepare 100 µL aliquots and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume Biotin 0.2 M 0.488 g NaOH (1 M) n/a Add to dissolve biotin ddH2O n/a Make up to 10 mL Total n/a 10 mL Terrific broth containing 50 µg/mL spectinomycin and 50 µg/mL carbenicillin
Prepare just before use in a safety cabinet.
Reagent Final concentration Quantity or Volume Terrific broth (Recipe 30) n/a 998 mL Spectinomycin (Recipe 42) 50 µg/mL 1 mL Carbenicillin (Recipe 43) 50 µg/mL 1 mL Total n/a 1 000 mL 1 mg/mL streptavidin Alexa FluorTM 488 conjugate
Protect from light, prepare 10 µL aliquots, and store at -20 °C. Stable for six months.
Reagent Final concentration Quantity or Volume Streptavidin Alexa FluorTM 488 conjugate 1 mg/mL 1 mg PBS (Recipe 3) 1× 1 mL Total n/a 1 mL 50 µg/mL streptavidin Alexa FluorTM 488 conjugate
Protect from light and prepare just before use.
Reagent Final concentration Quantity or Volume Streptavidin Alexa FluorTM 488 conjugate (1 mg/mL) 50 µg/mL 1 µL PBS (Recipe 3) 1× 19 µL Total n/a 20 µL 500 mM TCEP, pH 8.0
Store at 4 °C. Stable for at least one month.
Reagent Final concentration Quantity or Volume TCEP 500 mM 1.433 g ddH2O n/a 10 mL Adjust to pH 8.0 Total n/a 10 mL 1 mM TCEP, pH 8.0
Prepare just before use.
Reagent Final concentration Quantity or Volume TCEP (Recipe 49) 1 mM 2 µL ddH2O n/a 998 µL Total n/a 1 mL 10 mg/mL Alexa FluorTM 647 C2-Maleimide
Protect from light and store at -20 °C. Stable for at least one year.
Reagent Final concentration Quantity or Volume Alexa FluorTM 647 C2-Maleimide 10 mg/mL 1 mg DMSO 1× 500 µL Total n/a 500 µL DMEM medium without phenol red, 10% (v/v) FBS, 1× GlutaMAXTM, 1× penicillin-streptomycin
Store at 4 °C. Stable for at least one month.
Reagent Final concentration Quantity or Volume DMEM medium n/a 88 mL FBS 10% (v/v) 10 mL GlutaMAXTM (100×) 1× 1 mL Penicillin-streptomycin (100×) 1× 1 mL Total n/a 100 mL Confocal blocking buffer
Store at 4 °C. Use within one month.
Reagent Final concentration Quantity or Volume PBS (Recipe 3) 1× 10 mL BSA 5% (w/v) 0.5 g Triton X-100 0.25% (v/v) 25 µL Total n/a 10 mL Confocal dilution buffer
Store at 4 °C. Use within one month.
Reagent Final concentration Quantity or Volume PBS (Recipe 3) 1× 10 mL BSA 1% (w/v) 0.1 g Triton X-100 0.25% (v/v) 25 µL Total n/a 10 mL
Laboratory supplies
Vacuette® tube 4 mL CAT serum clot activator blood collection tubes (Greiner, Bio-one, catalog number: 454204)
Vacuette® tube 9 mL NH sodium heparin blood collection tubes (Greiner, Bio-one, catalog number: 455051)
Microcentrifuge tubes, 0.5 mL, 1.5 mL, 2 mL (Greiner, Bio-one, catalog numbers: 667201, 616201, 623201)
1.5 mL microcentrifuge tubes, RNase free (SARSTEDT, catalog number: 72.706.700)
PCR tubes and caps (VWR, catalog number: 20170-010)
50 mL conical centrifuge tubes (Greiner Bio-One, catalog number: 227261)
Serological pipettes, 2 mL, 5 mL, 10 mL, 50 mL (Greiner Bio-One, catalog numbers: 710180, 606180, 607180, 760180, 768160)
Screw cap microtubes (SARSTEDT, catalog number: 72.694.305)
MicrolanceTM 3 needles, 21G (BD, catalog number: 304432)
2.5 mL syringe (Greiner Bio-One, catalog number: SYR2)
8.5 cm Petri dishes (Greiner, Bio-one, catalog number: 633181)
Pipette tips, filtered, 20 µL, 200 µL, 1,000 µL (Mettler Toledo, Ranin, catalog numbers: 17005861, 17005863, 30389211)
Gene Pulser electroporation cuvettes, 0.1 cm gap (Bio-Rad, catalog number: 1652083)
Polystyrene semi micro cuvettes (Fisherbrand, catalog number: FB55147)
125 mL baffled flasks (VWR, catalog number: 214-0458)
250 mL baffled flasks (VWR, catalog number: 214-0460)
1 L baffled flasks (VWR, catalog number: 214-0464)
Micro quartz absorption cuvettes (Merck, Hellma®, catalog number: Z600210)
Square bioassay dish (Nunc, catalog number: 240845)
Reagent reservoirs, non-sterile (VWR, catalog number: 613-1176)
Reagent reservoirs, sterile (Axygen, catalog number: RES-V-25-S)
Deep-well 96-well 2 mL plate (Fisherbrand, catalog number: 11391555)
Deep-well 96-well 1 mL plate (Greiner, Bio-one, catalog number: 780215)
Deep-well 96-well 0.5 mL plate (Greiner, Bio-one, catalog number: 786261)
Cell culture adhesive seal (Azenta Life Sciences, catalog number: 4ti-0517)
Adhesive film for ELISA and general incubation (VWR, catalog number: 60941-062)
PCR foil seal (Azenta Life Sciences, catalog number: 4ti-0550)
Rigid semi-skirted 96-well PCR plate (Thermo Scientific, catalog number: AB-0990)
Framestar® 96-well skirted PCR plate (Azenta Life Sciences, catalog number: 4ti-0960)
96-well ELISA microplate (Greiner Bio-One, catalog number: 655061)
Octet SA biosensors (Sartorius, catalog number: 18-5019)
96-well black plate, polypropylene (Greiner Bio-One, catalog number: 655209)
96-well microtitre plate (Greiner Bio-One, catalog number: 65101)
250 mL PPCO centrifuge tube (Nalgene, catalog number: 3141-0250)
25 mL high speed PPCO centrifuge tube (Nalgene, catalog number: 3119-0010)
NuPage 4%–12% bis tris precast gel (Invitrogen, catalog number: NP0323BOX). A suitable alternative is homemade 12% running and 4% stacking SDS-PAGE gel
0.22 μm syringe filters (Agilent, catalog number: 5190-5116)
µ-Slide 8-well high polymer bottom chambered coverslip (ibidi, catalog number: 80807)
Microscope slides with ground edges (Fisherbrand, catalog number: 11572203)
Microscope coverslip, 12 mm diameter, no. 1.5 (Scientific Laboratory supplies, catalog number: MIC3334)
6-well cell culture dish (Greiner Bio-One, catalog number: 657160)
Dumont tweezer style 5 (Electron Microscopy Sciences, catalog number: 0203-5-PS)
Equipment
Pipetboy acu 2 pipette controller (Ingetra, catalog number: I155017)
Single-channel pipettes starter kit (Mettler Toledo, Ranin, catalog number: 30386738)
Multichannel pipettes 1–10 µL, 20–200 µL, 100–1,200 µL (Mettler Toledo, Ranin, catalog numbers: 17013802, 17013805, 17014496)
Microplate washer (Thermo Fisher, WellwashTM, catalog number: 5165000). If there is no well washer available, the wells can be washed manually using a multichannel pipette or using a wash bottle filled with PBST
CLARIOstar plus plate reader (BMG Labtech, catalog number: 430-501S-FL)
Class 2 microbiological safety cabinet (referred to as a safety cabinet and known as a laminar flow) (Contained Air solutions, catalog number: BioMAT 2). If no safety cabinet is available, working by a Bunsen burner is a suitable alternative. Working by an open flame with flammable EtOH should be done with caution
PCR workstation cabinet (Bigneat, catalog number: MW520-20)
NanoDropTM One/OneC Microvolume UV-Vis spectrophotometer (Thermo Scientific, catalog number: ND-ONE-W)
DynaMagTM 96-side magnet (Invitrogen, catalog number: 12331D)
PCR thermal cycler (Applied Biosystems, VeritiTM, catalog number: 4375305)
Eporator (Eppendorf, catalog number: 4309000027)
ChemiDocTM imaging system (Bio-Rad, catalog number: 12003154)
Cell density meter (Fisherbrand, catalog number: A0)
Multi Sub Electrophoresis System (Flowgen, catalog number: FMMS10)
SDS-PAGE equipment, mini gel tank (Invitogen, catalog number: A25977)
PowerPac basic power supply (Bio-Rad, catalog number: 1645050EDU)
JB Nova water bath (Grant, catalog number: JBN5)
Dry heating block (Grant, catalog number: QBD2)
Dual LED Blue/White Light Transilluminator (Thermo Fisher, catalog number: LB0100)
Safe ImagerTM viewing glasses (Thermo Fisher, catalog number: S37103)
HulaMixerTM sample mixer (Invitrogen, catalog number: 15920D). Agitation using this sample mixer is performed using the following settings: orbital = 5, rpm = 1
DynaMagTM-2 magnet (Invitrogen, catalog number: 12321D)
LSETM digital microplate shaker (Corning, catalog number: 4782-4). Agitation using the microplate shaker is performed at 500 rpm
Multifuge X4 Pro-MD (Thermo Scientific, catalog number: 75009500). In all centrifugation steps, maximum acceleration and deceleration rates are used unless otherwise specified
FrescoTM 21 microcentrifuge (Thermo Scientific, catalog number: 75002555)
Orbital shaking incubator (Shel Lab, catalog number: SI6/SI6R) (for 600 rpm agitation steps)
44R incubator shaker (New Brunswick, Innova®, catalog number: M1282-0006) (for 200 and 250 rpm agitation steps)
Octet® R8 (Sartorius, catalog number: 30-0518)
SP8 Lightning confocal microscope (Leica Microsystems Ltd.)
HeracellTM VIOS 160i CO2 Incubator (Thermo Scientific, catalog number: 51033559)
StuartTM SSM3 Gyratory Rocker (Cole-Parmer, catalog number: 51900-26)
Software and datasets
SnapGene (version 7.1.0, 2023, released 28 November 2023); requires a license
Octet® Analysis Studio Software (version 12.2.2.26); requires a license
LasX software for confocal microscope (version 5.2.1); requires a license
Procedure
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文章信息
版权信息
© 2024 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Eyssen, L. E. A., Ramadurai, S., Abdelkarim, S., Buckle, I., Cornish, K., Lin, H., Jones, A. K., Stephens, G. J. and Owens, R. J. (2024). From Llama to Nanobody: A Streamlined Workflow for the Generation of Functionalised VHHs. Bio-protocol 14(6): e4962. DOI: 10.21769/BioProtoc.4962.
Download Citation in RIS Format
分类
生物工程
生物化学 > 蛋白质 > 表达
微生物学 > 微生物生物化学 > 蛋白质 > 分离和纯化
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