All cloning reagents were obtained from New England Biolab (Gibson assembly Master Mix, Q5-mutagenesis kit) or Thermo Scientific (Fast Digest restriction enzymes). Reagents and building blocks for the solid phase peptide synthesis (SPPS) were from Anaspec, P3Bio, ChemPep, or Aaptec. The Chem Matrix Rink Amide resin was from Sigma. Solvents for SPPS, HPLC, and LC/MS, buffering agents, and additives were from Sigma or Fisher Scientific. Antibiotics, IPTG, DTT, and protease inhibitors (leupeptin, pepstatin, aprotinin, AEBSF) were from Gold Bio. KRX E.coli cells, NanoBit, and NanoGlow luciferase assay components were from Promega, and L-rhamnose was from Chem-Implex. tRNA from yeast was purchased from Sigma. Corning 384 Well Low Volume Black Round Bottom Polystyrene NBS Microplates were from Sigma. Anti-Histone H3 (acetyl K18) antibody - ChIP Grade (ab1191) was from Abcam, Anti-Histone H3 (acetyl K9) antibody (ab4441) was from Abcam. Secondary antibodies IRDye 680RD anti-mouse and IRDye 800CW anti-rabbit were from Li-Cor.
Nanoglow luciferase assay
Manufactured by Promega
NanoGlow is a luciferase assay product from Promega. It is designed for sensitive detection of luciferase activity in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pepstatin, by GoldBio (1 mentions)
Aprotinin, by GoldBio (1 mentions)
Fastdigest restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Anti mouse irdye 680rd, by LI COR (1 mentions)
Leupeptin, by GoldBio (1 mentions)
E coli krx cells, by Promega (1 mentions)
Anti rabbit irdye 800cw, by LI COR (1 mentions)
Nanobit, by Promega (1 mentions)
2 protocols using nanoglow luciferase assay
1
Peptide Synthesis and Epigenetic Assays
2
EV Immobilization and Recovery Assay
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Immobilization of EVs to white streptavidin-coated 96-well plates (PIERCE 15502) was performed in 100 µL of 25 mm HEPES pH 7.2, 150 mm NaCl, 0.05% Tween-20 either through SORT-enabled direct biotinylation of EVs or via biotinylated EV binding molecules (see EV affinity purification). After immobilization overnight at 4 °C, the plates were washed three times with 200 µL 25 mm HEPES pH 7.2, 150 mm NaCl, 0.05% Tween-20, and EV recovery was measured by NanoGlow luciferase assay (Promega N1120) and recovery normalized to the respective input signal.
For the analysis of luciferase recovery from prelysed EVs, concentrated EVs were incubated 25 mm HEPES pH 7.2, 150 mm NaCl, 0.5% NP-40 for 30 min at RT, before dilution to assay conditions.
Biotinylation of EVs for SIEVE was performed with concentrated EVs, and labeled EVs were diluted for binding to adjust for biotin binding capacity of the plates.
For the analysis of luciferase recovery from prelysed EVs, concentrated EVs were incubated 25 m
Biotinylation of EVs for SIEVE was performed with concentrated EVs, and labeled EVs were diluted for binding to adjust for biotin binding capacity of the plates.
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