5′-N-terminally labeled DNA oligonucleotides (anti-handles; Integrated DNA Technologies) were resuspended in Milli-Q ultrapure water at 2 mM concentration. The crosslinker benzylguanine (BG)-GLA-NHS (New England Biolabs) was dissolved in DMSO at 20 mM. N-terminally-labeled anti-handles and BG-GLA-NHS were mixed in a 1:3 ratio in a 67 mM HEPES, pH 8.5, buffer, and incubated at room temperature for 1 h. BG-DNA was then purified from excess crosslinkers by ethanol precipitation. The BG-DNA pellets were dried and stored at −20 °C until use.
Bg gla nhs
Manufactured by New England Biolabs
Sourced in United States
The BG-GLA-NHS is a heterobifunctional crosslinker that contains a benzylguanine (BG) group and an N-hydroxysuccinimide (NHS) ester group. The BG group reacts with SNAP-tag fusion proteins, while the NHS ester group reacts with primary amines on target molecules. This crosslinker can be used to covalently attach SNAP-tag fusion proteins to amine-containing surfaces or molecules.
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Lab products found in correlation
Zeba spin desalting column, by Thermo Fisher Scientific (2 mentions)
Illustra g 50, by GE Healthcare (2 mentions)
Carboxy latex beads, by Thermo Fisher Scientific (2 mentions)
Amine coated qds, by Thermo Fisher Scientific (2 mentions)
Rabbit polyclonal αgfp antibodies, by Fortrea (2 mentions)
Btsci, by New England Biolabs (1 mentions)
Quantity one 1 d analysis software, by Bio-Rad (1 mentions)
αcd28, by BioLegend (1 mentions)
α cd4, by BioLegend (1 mentions)
αcd19, by BioLegend (1 mentions)
Megacd40l, by Enzo Life Sciences (1 mentions)
Centrifugal filter device, by Merck Group (1 mentions)
Rituxan, by Genentech (1 mentions)
Herceptin, by Genentech (1 mentions)
Nanodrop onec, by Thermo Fisher Scientific (1 mentions)
Microtoflc mass spectrometer, by Bruker (1 mentions)
Eurospher 2 100 5 c18 column, by Knauer (1 mentions)
13 protocols using bg gla nhs
1
Preparation of BG-Labeled DNA Oligonucleotides
2
BG-DNA Conjugation Protocol
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DNA anti-handles (5'-labeled amino-DNA oligonucleotides) were resuspended in deionized H2O at 2 mM. BG-GLA-NHS (New England BioLabs) was dissolved in DMSO at 20 mM. DNA anti-handles were then mixed with BG-GLA-NHS in a 1:3 volumetric ratio in 70 mM HEPES buffer (pH 8.5) and incubated at room temperature (r.t.) for 1 hour. The BG-DNA product was then purified from excess BG-GLA-NHS by ethanol precipitation. Dried BG-DNA pellets were stored at -20°C until use.
3
Preparation of Biotinylated DNA Tethers from M13 Phage
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Circular ssDNA from the M13 bacteriophage (New England Biolabs) was linearized at a single site using the restriction enzyme BtscI (New England Biolabs) and a site-specific oligonucleotide93 (link). A forward primer containing dual 5′ biotins (Integrated DNA Technologies) was purchased and a reverse 5′ benzylguanine primer was synthesized from a primer with a 5′ primary amine on a 12-carbon linker (IDT) (Supplementary Table 2 ). The synthesis was accomplished with excess BG-GLA-NHS (NEB) in PBS pH 7.4 for 1 h at room temperature and buffer exchanged on a Zeba 7k MWCO desalting column. The forward and reverse primers were used to amplify a 2385 base pair DNA fragment from the linearized M13 template using Q5 DNA Polymerase 2× HotStart Master Mix (NEB). The amplified DNA tether was purified using Ampure XP beads at a ratio of 0.7:1 suspended beads to PCR mix and eluted in TBS + Ca2+ buffer. To minimize damage to the DNA tethers, appropriate techniques were used to minimize unnecessary hydrodynamic stresses, and DNAse-free lab supplies were used to minimize potential degradation. The quality of the DNA tethers was checked with gel electrophoresis, and the tethers were stored at −20 °C in aliquots until use.
4
DNA Oligo Functionalization for SNAP Tag Binding
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Complementary amine-modified 63 bp DNA oligos (IDT) were used. The sequences were /5AmMC12/GT CAA TAA TAC GAT AGA GAT GGC AGA AGG GAG AGG AGT AGT GGA GGT AGA GTC AGG GCG AGA T (kinesin oligo) and /5AmMC12/AT CTC GCC CTG ACT CTA CCT CCA CTA CTC CTC TCC CTT CTG CCA TCT CTA TCG TAT TAT TGA C (GBP oligo). These oligo designs were adapted from previous work (Belyy et al., 2016 (link)) and confirmed to have a low probability of forming secondary structures. To functionalize the oligos with BG for SNAP tag binding, 250 μM of each oligo was incubated with 13.28 mM of BG-GLA-NHS (NEB) in 100 mM sodium borate and 50% v/v DMSO. The reaction was then desalted into ×1 PBS supplemented with 1 mM DTT using a PD MiniTrap column (Cytiva, Marlborough, MA). The BG-labeling was confirmed using a 10% TBE-Urea electrophoresis gel, and the BG-oligo concentration was determined via absorbance at 260 nm.
5
Quantifying Shiga Toxin Transport
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Stx was labeled with the SNAP-tag substrate BG-GLA-NHS (New England BioLabs) according to the manufacturer's instructions and using 3:1 ratio BG to toxin. Stx1-mut-BG was then labeled with iodine as described in [13 (link)]. HEp-2 cells stably expressing GalT-GFP-SNAP or ER-GFP-SNAP were treated with 1 mM FDG for 4 h or with 2 μg/ml brefeldin A (BFA) for 30 min in complete growth medium. To analyze Golgi transport, HEp-2-GalT-GFP-SNAP cells were incubated with 50 ng/ml 125I-Stx1-mut-BG for 1 h in the presence of the drugs. For Stx transport to the ER, Hep-2-ER-GFP-SNAP cells were incubated with 50 ng/ml 125I-Stx1-mut-BG for 5 h. Then the cells were lysed, and Stx-GalT-GFP-SNAP or Stx-ER-GFP-SNAP (later referred to as Stx-SNAP) was immunoprecipitated from the lysates by GFP-trap (ChromoTek GmbH) and run on SDS-PAGE. Proteins were transferred to PVDF membranes, visualized by autoradiography and quantified using the Quantity One 1-D Analysis Software (Bio-Rad Laboratories). Both Stx subunits, A and B, were coupled to the SNAP-tag proteins, and gave essentially similar quantitative data, although the signal for 125I-StxB-SNAP was brighter, and thus was used for the final quantification.
6
Antibody and Protein Labeling Protocol
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Antibodies were desalted using Zebaspin Desalting Columns (7 kDa MWCO, Thermo Fisher Scientific, Cat. # 89882) and diluted to a concentration of 0.5 mg/ml, before incubation with a 40-fold molar excess of BG-GLA-NHS (New England Biolabs, Cat. # S9151S). To test different ratios of BG-GLA-NHS, 10-, 20-, 40-, or 80-fold molar excess of BG-GLA-NHS was added to the antibodies, The reaction was allowed to continue for 30 min at room temperature before it was stopped by removing the unreacted NHS-BG substrate using Zebaspin Desalting Columns (7 kDa MWCO). MegaCD40L (Enzo Lifesciences, Cat. # ALX-522-110-C010) was labeled with a 10-fold molar excess of NHS-GLA-BG, due to the high number of surface exposed lysines. αCD3 (BioLegend Cat. # 317302), αCD4 (BioLegend Cat. # 317402) and αCD19 (BioLegend Cat. # 302202) were each labeled with a 40-fold molar excess of NHS-GLA-BG. αCD28 (BioLegend Cat. # 302933) was labeled with a 20-fold molar excess of NHS-GLA-BG, due to significant precipitation at 40-fold molar excess. All BG-labeled antibodies were resuspended in PBS, and their concentration was determined using the IgG function on a Nanodrop 2000 device.
7
Labeling CARDS Toxin with BG-GLA-NHS
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BG-GLA-NHS (New England Biolabs) was dissolved in dimethyl sulfoxide and added to WT or K3 CARDS toxin to a final concentration of 1 mM (labeling ratio, 1:20). The reaction mixtures were mixed well for 1 h at RT, and unbound BG-GLA-NHS was quenched with 100 mM Tris HCl (pH 8.0) for 15 min and washed four times with 1× PBS, using a centrifugal filter device (Millipore) with a cutoff of 3,000 nominal molecular weight limit.
8
Covalent Labeling of Oligonucleotides
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Benzyl-guanine NHS ester (BG-GLA-NHS; NEB, Ipswich, MA) was covalently linked to the C6-amine modified oligonucleotides (BG-oligo 1 and BG-oligo 5; Supplementary file 1 ). Briefly, 0.17 mM C6-amine-oligo-Cy3 was incubated with 11.6 mM BG-GLA-NHS in 0.1 M NaBO3 for 2–4 hr at 37°C with shaking. BG-labeled oligo was purified twice through Illustra G-50 micro columns (GE Healthcare, Pittsburgh, PA) equilibrated in 2 mM Tris, pH 8.5. BG-oligo concentration was determined from absorbance at 260 nm.
9
Antibody Labeling with BG-GLA-NHS
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Rituximab (Rituxan, Genentech), Cetuximab (Erbitux, Eli Lily), Trastuzumab (Herceptin, Genentech) and FMC63 (Novus Biologicals) underwent buffer exchange into PBS using 2 mL 7 K MWCO Zeba Spin Desalting Columns (ThermoFisher Scientific). The Rituximab Fab fragment was generated using the Fab Preparation Kit (Pierce) following the manufacturer’s protocol. Antibodies were then co-incubated with a 20-fold molar excess of BG-GLA-NHS (NEB) for 30 minutes at room temperature, followed by buffer exchange into PBS using 2 mL 7 K MWCO Zeba Spin Desalting Columns.
10
DNA Nanotubes for Protein Binding
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Cy5-labeled 10-helix DNA nanotubes composed of 40 single-stranded DNA tiles with 14- or 28-nm spacing between single-stranded protein binding handles were prepared with biotin strands for surface attachment using an annealing protocol as previously described (34 (link)). The nanotube DNA handles at 14 or 28 nm are designed to anneal to DNA strands bound to a SNAP protein encoded with the GFP nanobody. The SNAP protein binds benzyl-guanine-treated DNA oligos. To prepare the benzyl-guanine oligo, benzyl-guanine NHS ester (BG-GLA-NHS; New England Biolabs, Ipswich, MA) was covalently linked to C6-amine oligonucleotides (with or without Cy3 modification, C6-amine-Cy3-a′ or C6-amine-a′). This was accomplished by incubating 0.17 mM C6-amine-a′ or C6-amine-b′ with 11.6 mM BG-GLA-NHS in 0.1 M sodium borate, pH 8.5 at 37°C for ∼4 h with rotation. The BG-labeled oligonucleotide was then purified twice through Illustra G-50 micro columns (GE Healthcare, Buckinghamshire, UK) equilibrated in 2 mM Tris, pH 8.5, since the primary amine reacts with any unreacted benzyl-guanine. The final BG-labeled oligonucleotide concentration was determined by measuring Cy3 absorbance (for BG-Cy3-a′ or BG-Cy3-b′) or by estimating the concentration of single-stranded DNA (for BG-a′ or BG-b′) using a NanoDrop spectrophotometer (NanoDrop OneC, Thermo Fisher, Waltham, MA).
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