Recombinant SARS-CoV-2 RBD (from the Wuhan-1 strain, which shares an identical S protein to the WA-1 strain) was generated as previously described (25 (link)). For tetramer generation, RBD proteins were biotinylated with the BirA500 kit (Avidity), tetramerized with streptavidin-phycoerythrin (SA-PE) (Agilent, PJRS301-1), and stored in 50% glycerol at –20°C as previously described (45 (link)). Decoy reagents were generated by tetramerizing an irrelevant biotinylated protein with SA-PE previously conjugated to Dylight594 NHS Ester (Thermo Fisher Scientific, 46413) and Dylight650 NHS Ester (Thermo Fisher Scientific, 62266).
Dylight594 nhs ester
Manufactured by Thermo Fisher Scientific
Sourced in United States
DyLight594 NHS Ester is a fluorescent labeling reagent. It is an N-Hydroxysuccinimide (NHS) ester derivative of the DyLight594 dye. The DyLight594 dye has an excitation maximum at 593 nm and an emission maximum at 618 nm.
Automatically generated - may contain errors
Lab products found in correlation
Dylight650 nhs ester, by Thermo Fisher Scientific (2 mentions)
Bira500 kit, by Avidity (2 mentions)
Sa pe, by Agilent Technologies (1 mentions)
Sa apc, by Thermo Fisher Scientific (1 mentions)
Dylight 755 nhs ester, by Thermo Fisher Scientific (1 mentions)
Dylight 594, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 2 centrifugal filter device, by Merck Group (1 mentions)
Alexa 488 c5 maleimide, by Thermo Fisher Scientific (1 mentions)
Pluronics f 127, by Merck Group (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Axiovision, by Zeiss (1 mentions)
Acridine orange, by Thermo Fisher Scientific (1 mentions)
Dq green bsa, by Thermo Fisher Scientific (1 mentions)
Dq red bsa, by Thermo Fisher Scientific (1 mentions)
Bioactive compound library l4000, by Targetmol (1 mentions)
Malachite green phosphate assay kit, by Merck Group (1 mentions)
8 protocols using dylight594 nhs ester
1
Recombinant SARS-CoV-2 RBD Tetramerization
2
Recombinant SARS-CoV-2 RBD Production and Tetramerization
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Recombinant SARS-CoV-2 RBD (Wuhan-1, Wu-1) was generated by standard transient transfection followed by IMAC purification as described previously (Walls et al., 2020 (link)). Recombinant SARS-CoV-2 RBD (B.1.1.351, β) was generated by transient transfection from the SARS-CoV-2-Beta-RBD-Avi (K417N-E484K-N501Y) construct synthesized by GenScript into CMVR with an N-terminal mu-phosphatase signal peptide and a C-terminal octa-histidine tag, flexible linker, and avi tag (GHHHHHHHHGGSSGLNDIFEAQKIEWHE) as described previously (Tortorici et al., 2021 (link)). For tetramer generation, RBD proteins were biotinylated with the BirA500 kit (Avidity), tetramerized with streptavidin-phycoerythrin (SA-PE) or streptavidin-allophycocyanin (SA-APC) (Agilent) and stored in 50% glycerol at -20oC as previously described (Krishnamurty et al., 2016 (link)). Decoy reagents were generated by tetramerizing an irrelevant biotinylated protein with SA-PE previously conjugated to Dylight594 NHS Ester (ThermoFisher) and Dylight650 NHS Ester (ThermoFisher) or SA-APC previously conjugated to Dylight755 NHS Ester (ThermoFisher).
3
Chemokine Conjugation and Purification
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Murine CCL19 was purchased from R&D systems, USA. Biotinylated, truncated murine CCL21 (mCCL21 24–98 bio) was synthesized by ALMAC (Craigavon, UK). Chemokines were reconstituted as follows: Desiccated chemokines were reconstituted to a concentration of 25 μg/mL in PBS and stored at −20 °C. Prior to use, mCCL21 24–98 bio was diluted to a working concentration of 250 ng/mL in PBS.
DyLight 594 (Thermo Fisher Scientific) labeled mCCL21 24–98 bio was prepared following the manufacturers protocol. Briefly, 100 μg mCCL21 24–98 bio were reconstituted in 100 μL phosphate buffer containing 0.1 M Na2HPO4, 0.15 M NaCl adjusted to pH 7.2–7.5. 65 μg DyLight 594 NHS ester (Thermo Fisher Scientific) were added and the mixture was allowed to react for 1 h at room temperature. Subsequently, excessive DyLight 594 NHS ester was quenched for 1 h at room temperature by adding 1.4 mL of Tris/HCl pH 7.6. mCCL21 24–98 bio DL595 was purified using MW10 kDa spin columns (Amicon Ultra-2 Centrifugal Filter devices, Millipore) and stored at −20 °C.
DyLight 594 (Thermo Fisher Scientific) labeled mCCL21 24–98 bio was prepared following the manufacturers protocol. Briefly, 100 μg mCCL21 24–98 bio were reconstituted in 100 μL phosphate buffer containing 0.1 M Na2HPO4, 0.15 M NaCl adjusted to pH 7.2–7.5. 65 μg DyLight 594 NHS ester (Thermo Fisher Scientific) were added and the mixture was allowed to react for 1 h at room temperature. Subsequently, excessive DyLight 594 NHS ester was quenched for 1 h at room temperature by adding 1.4 mL of Tris/HCl pH 7.6. mCCL21 24–98 bio DL595 was purified using MW10 kDa spin columns (Amicon Ultra-2 Centrifugal Filter devices, Millipore) and stored at −20 °C.
4
Fluorescent Bacterial Labeling Protocol
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For fluorescent labeling with Dylight 594 NHS Ester (Thermo Fisher, Waltham, USA), bacteria, suspended in sterilized eggwater (see above), were spun down and resuspended in 0.1 M Na2CO3 (pH 8.3–9.0). Bacteria were subsequently spun down a second time, and incubated in 300 μl 0.1 M Na2CO3 (pH 8.3–9.0) containing 6.5 μl of Dylight dye (10 mg/ml in DMSO) at room temperature for 2 h, shielded from light and with gentle agitation. After incubation with the dye, stained bacteria were washed twice with autoclaved egg water and resuspended at a final A600 optical density of 0.005 which was used directly as colonization medium added to 3 DPF embryos. Staining had no apparent effect on bacterial viability or on the larvae. 2 days later, at 5 DPF, larvae were washed briefly in autoclaved egg water and kept in autoclaved egg water for an additional 2 h, after which they were imaged by confocal microscopy. Patterns have been validated independently in triplicate.
5
Affinity Purification of Anti-Mouse COL7 IgG
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Affinity purified rabbit anti-mouse COL7 IgG was produced as described (10 (link)). Briefly, rabbits were immunized with an immunodominant fragment of murine COL7. Total immune IgG was isolated from rabbit serum using protein G, followed by isolation of COL7-specific IgG using COL7 coupled to an Affi-Gel 10 matrix (Bio-Rad #1536099). COL7 affinity purified IgG was labeled with DyLight™ 594 NHS ester (Thermo Scientific #46412) according to manufactures instructions. Monoclonal FcγRIV blocking antibody 9E9 was kindly provided by Falk Nimmerjahn (9 (link)), the isotype control IgG1κ (clone A19-3) was purchased from BD Biosciences (#553968).
6
Quantification of HP1α-Histone Interactions
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Recombinantly expressed and purified HP1α (MW = 22kDa) was purchased (lyophilized against PBS buffer) from Biomatik Corporation (www.biomatik.com ) (Ontario, Canada). Unmodified and methylated histone H3 peptides (1 (link)–22 ) (MW =2354) were purchased in lyophilized form from Bachem Americas,Inc (Torrance, CA) (Bachem.com ) and used as received. Chemicals including Alexa-488 C5-Maleimide, Dylight-594 NHS ester, Nucleic acid dimer sampler kit (YOYO-3 iodide, POPO-1 iodide), dsDNA (NoLimits 2.5kbp, 4kbp, 10kbp) as well as dialysis cassettes (Slide-A-Lyzer G2 3kda cutoff) were obtained from Thermo Fisher Scientific (Waltham, MA). Potassium chloride, Pluronics F127 was obtained from Sigma Aldrich (St. Louis, MO). HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)) was obtained from Fisher Scientific (Pittsburg, PA). Peptides were reconstituted at 25 mg ml−1 in nuclease-free ultra pure water as per the manufacturer’s instructions and stored as aliquots at −20°.HP1α was reconstituted (0.5 mg ml−1) in nuclease-free ultra pure water as per the manufacturer’s instructions and stored (with 10% glycerol) as flash-frozen 200 ul aliquots in −80°C.
7
Imaging Gonococcal Infection Dynamics
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VK2/E6E7 were grown in 35 mm poly-D-lysine–coated glass bottom dishes. Prior to infection, N. gonorrhoeae MS11 P+ were stained with DyLight 594 NHS Ester (Thermo Scientific, Waltham, MA, USA) for 7 min at 37°C in 5% CO2. Cells were then infected with stained N. gonorrhoeae for 10 h, stained with Hoechst 33342, (0.5 µg/ml) for 30 min at 37°C in 5% CO2, and washed twice with PBS. The dish was transferred to a live-cell incubator that was connected to an inverted microscope. Cells were maintained at 37°C in 5% CO2 throughout the microscopy analysis and monitored for additional 12 h to capture cells undergoing mitosis. Images of mitotic cells were taken through a 100x oil objective in 40–60 z-stacks with optimized optical thickness. Images were further deconvolved by an iteration algorithm and further processed by Inside 4D software Axiovision (Carl Zeiss, GmbH, Göttingen, Germany) and Photoshop CS5 (Adobe).
8
Recombinant Amyloid-beta Peptide Assay
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Recombinant human Aβ1-42 peptides (A-1163), and synthetic fluorescein-labeled Aβ1-42 (A-1119) were from rPeptide; DQ-red-BSA (D12051), and DQ-green-BSA (D12050) were from Invitrogen; Dextran, Cascade Blue, 10,000 MW (D1976), and DyLight 594 NHS Ester (46,413) were from Thermo Scientific; acridine orange (A6014), Resomer® RG 503 H, poly(D,L-lactide-co-glycolide) (acidic nanoparticle, 719870), hyaluronic acid sodium salt from Streptococcus equi MW 8,000–15,000 (LMWHA, 40583), hyaluronic acid sodium salt from Streptococcus equi MW 1,750,000–2,000,000 (HMWHA, 02737), and Malachite Green Phosphate Assay Kit (MAK307) were from Sigma-Aldrich; Bioactive compound library (L4000) was from TargetMol; Lipofector-pMAX (AB-LF-M100) was from AptaBio; siMapt (17762–1 and 17762–2)was from Bioneers; HYAL1 lentiviral particle was from VectorBuilder.
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