From 5ae475dffb8d55feca42201fec92ca8e16ce2876 Mon Sep 17 00:00:00 2001
From: =?UTF-8?q?Aileen=20Kr=C3=BCger?= <ai.krueger@fz-juelich.de>
Date: Mon, 5 May 2025 16:43:12 +0200
Subject: [PATCH] EMSAs
---
.gitattributes | 10 ++++++++++
.../README.md | 0
.../dataset/.gitkeep | 0
.../dataset/EMSA\342\200\230s DtxR.pptx" | 3 +++
.../dataset/Gel2.tiff | 3 +++
.../dataset/Gel3.tiff | 3 +++
.../dataset/Gel4.tiff | 3 +++
.../dataset/Gel5.tiff | 3 +++
.../dataset/Gel6.tiff | 3 +++
.../dataset/controlgel1.tiff | 3 +++
.../dataset/controlgel2.tiff | 3 +++
.../isa.assay.xlsx | Bin 0 -> 7146 bytes
.../protocols/.gitkeep | 0
.../protocols/EMSA's.md | 2 ++
studies/4_InVitroBindingOfDtxR/README.md | 0
studies/4_InVitroBindingOfDtxR/isa.study.xlsx | Bin 0 -> 14846 bytes
.../4_InVitroBindingOfDtxR/protocols/.gitkeep | 0
.../protocols/DNA preparation.md | 1 +
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.../4_InVitroBindingOfDtxR/resources/.gitkeep | 0
.../resources/20250224_EMSAPrimer.xlsx | Bin 0 -> 9454 bytes
.../NC_003450.3_peak regions marked.dna | 3 +++
22 files changed, 41 insertions(+)
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diff --git a/assays/4_ElectronMobilityShiftAssays_DtxR/protocols/.gitkeep b/assays/4_ElectronMobilityShiftAssays_DtxR/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/assays/4_ElectronMobilityShiftAssays_DtxR/protocols/EMSA's.md b/assays/4_ElectronMobilityShiftAssays_DtxR/protocols/EMSA's.md
new file mode 100644
index 0000000..e43bd38
--- /dev/null
+++ b/assays/4_ElectronMobilityShiftAssays_DtxR/protocols/EMSA's.md
@@ -0,0 +1,2 @@
+The purified DNA fragments of 121 bp size covering the region of identified ChAP-Seq peaks (61 bp peak extended 30 bp to each side; final concentration 31 nM) were incubated
+for 30 min at room temperature with 0, 50 or 200-fold purified DtxR protein excess or 0, 300 and 400-fold excess, respectively. The reaction buffer contained 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 40 mM KCl, 5% (v/v) glycerol, 1 mM dithiothreitol (DTT), and 150 μM MnCl2. Afterwards,separation followed by a gel electrophoresis using a native polyacrylamide (10%) gel supplemented with 1 mM DTT and 150 μM MnCl2. Electrophoresis was performed at 170 V using TB buffer (89 mM Tris, 89 mM boric acid) supplemented with 1 mM DTT and 150 μM MnCl2 for 1.5 h. Gels were stained for 20 min with ethidium bromide. 121 bp promoter region of acn served as negative control. Protocol was adjusted according to Wennerhold and Bott (2006).
\ No newline at end of file
diff --git a/studies/4_InVitroBindingOfDtxR/README.md b/studies/4_InVitroBindingOfDtxR/README.md
new file mode 100644
index 0000000..e69de29
diff --git a/studies/4_InVitroBindingOfDtxR/isa.study.xlsx b/studies/4_InVitroBindingOfDtxR/isa.study.xlsx
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diff --git a/studies/4_InVitroBindingOfDtxR/protocols/.gitkeep b/studies/4_InVitroBindingOfDtxR/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/studies/4_InVitroBindingOfDtxR/protocols/DNA preparation.md b/studies/4_InVitroBindingOfDtxR/protocols/DNA preparation.md
new file mode 100644
index 0000000..521debc
--- /dev/null
+++ b/studies/4_InVitroBindingOfDtxR/protocols/DNA preparation.md
@@ -0,0 +1 @@
+DNA fragments of 121 bp size covering the region of identified ChAP-Seq peaks were generated using respective primer pairs. Purification followed using NucleoSpin Gel and PCR Clean-up Kit (Macherey Nagel, Düren, Germany) eluting in H2O. Concentrations were measured using nanophotometer.
\ No newline at end of file
diff --git a/studies/4_InVitroBindingOfDtxR/protocols/Protein purification.md b/studies/4_InVitroBindingOfDtxR/protocols/Protein purification.md
new file mode 100644
index 0000000..79e1bea
--- /dev/null
+++ b/studies/4_InVitroBindingOfDtxR/protocols/Protein purification.md
@@ -0,0 +1 @@
+The expression plasmid pET24b-dtxR-C in E. coli BL21(DE3) was used for overproduction of DtxR of Corynebacterium glutamicum containing a His-Tag (HHHHLEHHHHHH) at the carboxyl terminus as previously described (Wennerhold and Bott, 2006). DtxR was purified using Ni-NTA agarose column material (Thermo Fisher Scientific, USA) according to manufacturer’s instructions to the gravity flow protocol. Fractions containing DtxR were pooled and elution buffer was exchanged against TG buffer (30 mM Tris-HCl, 10% (v/v) glycerol, pH 7.5) using PD-10 desalting columns (Cytiva, USA). Concentration was firstly evaluated with Bradford assay, and further measured using Qubit Protein Assay Kit (Invitrogen).
\ No newline at end of file
diff --git a/studies/4_InVitroBindingOfDtxR/resources/.gitkeep b/studies/4_InVitroBindingOfDtxR/resources/.gitkeep
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diff --git a/studies/4_InVitroBindingOfDtxR/resources/20250224_EMSAPrimer.xlsx b/studies/4_InVitroBindingOfDtxR/resources/20250224_EMSAPrimer.xlsx
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diff --git a/studies/4_InVitroBindingOfDtxR/resources/NC_003450.3_peak regions marked.dna b/studies/4_InVitroBindingOfDtxR/resources/NC_003450.3_peak regions marked.dna
new file mode 100644
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