A His-tagged Slp1 construct was made by cloning the N-terminus through the DNA binding domain of Slp1 (amino acids #1 through 216) into a pET14b vector. His-tagged Exd-Hth heterodimers, Abd-A, and Slp1 protein were purified from BL21 using Ni+ beads, as described previously [36 (link), 42 (link)]. SDS-PAGE and Coomassie blue staining was used to confirm purification of the protein of interest. For EMSA, fluorescent DNA probes (Integrated DNA Technologies) were mixed with proteins as indicated in Figure legends, incubated for 10 min prior to running on polyacrylamide gels, and imaged using an Odyssey LiCOR cLX scanner as previously described [37 (link), 43 (link)].
Fluorescent dna probes
Manufactured by Integrated DNA Technologies
Fluorescent DNA probes are synthetic oligonucleotides labeled with fluorescent dyes. They are used to detect and quantify specific DNA sequences in various molecular biology applications, such as real-time PCR, in situ hybridization, and microarray analysis.
Automatically generated - may contain errors
2 protocols using fluorescent dna probes
1
Purification and EMSA of Slp1, Exd-Hth, and Abd-A
2
Exosome Profiling with TPEX
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For TPEX measurement with antibodies, we isolated exosomes from various cell lines and incubated the samples with fluorescent antibodies (anti-CD63, BD Biosciences, and anti-CD24, eBioscience; 1 μg/ml). Without any purification, we added AuNP and gold salt mixture to this reaction, as described above, and measured the resultant changes in fluorescence. All measurements were compared against gold standard ELISA analysis using the same antibodies (see below for details).
For TPEX miRNA detection, whole exosomes were subjected to additional fixation and permeabilization (BD Biosciences), before being labeled with fluorescent DNA probes against miRNA targets (Integrated DNA Technologies; 10 μM). Without any purification, we added AuNP and gold salt mixture to this reaction, as described above, and measured the resultant changes in fluorescence. All measurements were compared against gold standard TaqMan assays (Thermo Fisher Scientific) through polymerase chain reaction (Applied Biosystems).
For TPEX miRNA detection, whole exosomes were subjected to additional fixation and permeabilization (BD Biosciences), before being labeled with fluorescent DNA probes against miRNA targets (Integrated DNA Technologies; 10 μM). Without any purification, we added AuNP and gold salt mixture to this reaction, as described above, and measured the resultant changes in fluorescence. All measurements were compared against gold standard TaqMan assays (Thermo Fisher Scientific) through polymerase chain reaction (Applied Biosystems).
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