Serum samples collected at necropsy were analyzed for anti‐dsDNA antibodies by ELISA as described earlier [49 (link), 53 (link)]. Briefly, pGEM‐3zf plasmid DNA (Promega) was digested with S1‐nuclease (Thermo Fisher Scientific) and biotinylated using Photoprobe Biotin (Vector Labs) following the manufacturer's instructions. Biotinylated dsDNA (1 ng/mL) was used for the assay.
Photoprobe biotin
Manufactured by Vector Laboratories
Sourced in United States
Photoprobe Biotin is a photoactive biotin reagent designed for protein labeling and detection applications. It provides a covalent attachment of biotin to proteins through a UV-induced reaction.
Automatically generated - may contain errors
Lab products found in correlation
S1 nuclease, by Thermo Fisher Scientific (3 mentions)
Pgem 3zf plasmid dna, by Promega (2 mentions)
Pierce streptavidin coated plates, by Thermo Fisher Scientific (1 mentions)
Opd solution, by Merck Group (1 mentions)
Accuprep genomic dna extraction kit, by Bioneer (1 mentions)
Dynabeads kilobasebinder kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using photoprobe biotin
1
Quantifying Anti-dsDNA Antibodies in Serum
2
Serum Anti-dsDNA Antibody ELISA Assay
3
Genomic DNA extraction and biotinylation
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Genomic DNA was extracted from 80% confluent dishes of MDA-MB-231 cells using the Accuprep Genomic DNA extraction Kit by Bioneer Corp (Daejeon, Republic of Korea) according to the manufacturer’s instructions. DNA was digested with Sac II, precipitated, and suspended in TE buffer and biotinylated by thermal coupling with photoprobe biotin (Vector Laboratories, Burlingame, CA, USA) per manufacturer’s instructions. Unlabeled biotin was removed and DNA was precipitated and resuspended in TE.
biotinylated DNA was pulled down by Dynabeads M-280 streptavidin using Dynabeads kilobaseBINDER kit (Invitrogen, Carlsbad, CA, USA) per manufacturer’s instructions. Briefly, the beads were incubated with DNA at room temperature with constant vertical shaking, washed and incubated with protein extract.
biotinylated DNA was pulled down by Dynabeads M-280 streptavidin using Dynabeads kilobaseBINDER kit (Invitrogen, Carlsbad, CA, USA) per manufacturer’s instructions. Briefly, the beads were incubated with DNA at room temperature with constant vertical shaking, washed and incubated with protein extract.
4
Genomic DNA Isolation and GC Content Analysis
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The procedure of Marmur [32 ] was followed to isolate high molecular weight genomic DNA for GC content analysis. By determining the mid-point (Tm) of the thermal denaturation curve of the given DNA [33 (link)] and by using the equation of Owen & Hill [34 ], the G+C base composition was calculated. DNA-DNA hybridization (DDH) was conducted using fluorometric direct binding method [35 ] and the hybridization experiments were performed in micro dilution wells. For labelling the DNA as probes, photoprobe biotin from Vector Laboratories, Inc., was used. The temperature used for DNA-DNA hybridization was 53°C.
5
Anti-dsDNA Antibody ELISA Protocol
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