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Methods Mol Biol. Author manuscript; available in PMC 2016 May 31. Published in final edited form as: Methods Mol Biol. 2015 ; 1334: 85–99. doi:10.1007/978-1-4939-2877-4_5.
Southwestern Blotting Assay Yinshan Jia, Linda Nagore, and Harry Jarrett
Abstract
Author Manuscript
Southwestern blotting is a technique used to study DNA-protein interactions. This method detects specific DNA-binding proteins by incubating radiolabeled DNA with a gel blot, washing, and visualizing through autoradiography. A blot resulting from 1-dimensional SDS-PAGE reveals the molecular weight of the binding proteins. To increase separation and determine isoelectric point a 2-dimensional gel can be blotted. Additional dimensions of electrophoresis, such as a gel shift (EMSA), can precede isoelectric focusing and SDS-PAGE to further improve separation. Combined with other techniques, such as mass spectrometry, the DNA-binding protein can be identified.
Keywords Transcription; Regulation; DNA; Response elements; Oligonucleotide; Promoter; Immunoblots; Nitrocellulose membrane; PVDF membrane; Southwestern blotting
Author Manuscript
1 Introduction Regulation of gene expression is essential in human development as well as pathogenesis [1]. The gene regulatory mechanism involves distinct 6–8 bp DNA motifs referred to as response elements, which bind transcription factors [2]. Transcription factors are DNAbinding proteins that interact with unique response elements at the promoter region of DNA, or other functional cisacting response elements, resulting in either gene expression or repression [1]. Identification of transcription factors specific to a particular gene is not only significant to gene regulation but also to understanding gene function. Southwestern blotting assay (SWB) is one of the most powerful techniques to explore protein-DNA interaction and transcription factor regulation.
Author Manuscript
SWB, similar to other blotting techniques, separates proteins (or DNA) by gel electrophoresis. The gel containing the separated proteins is electro-transferred (blotted) to a membrane, such as nitrocellulose (NC) or polyvinylidine difluoride (PVDF). To detect the DNA-binding proteins, the proteins are partially renatured and bound to nanomolar concentrations of radiolabeled DNA. Any unbound DNA is washed away, and then bands on the blot are detected by autoradiography. This technique is especially useful in the identification of transcription factor as it gives information on the molecular weights of all DNA-binding proteins involved with a particular sequence of DNA. An experimental procedure is shown schematically in Fig. 1.
Jia et al.
Page 2
Author Manuscript
Since the original SWB assay, first described in 1980 [3], many extensions of the SWB method have been developed [4, 5]. In this chapter, three detailed SWB methods are described that differ in the number of electrophoresis dimensions used for protein separation. 1D-SWB uses separation on a single SDS-PAGE dimension, 2D-SWB uses separation by isoelectric focusing followed by SDS-PAGE in the second dimension, while 3D-SWB uses separation by DNA gel shift on a non-denaturing gel before 2D-SWB. Coupled with proteomic techniques, several transcription factors have been identified [4]. Additionally, a method has been developed that allows multiple reprobes with different oligonucleotides on one blot [6]. Each of these SWB techniques provides alternatives to investigate regulatory transcription factors in vitro.
2 Materials Author Manuscript
2.1 Oligonucleotide Preparation 1.
Oligonucleotide: Approximately 20–25 nt in size along with its complementary (antisense) strand, containing one protein-binding motif (0.1 µmol).
2.
TE buffer: 10 mM Tris (free base), titrated to pH 7.5 with HCl, 1 mM EDTA.
3.
0.5 M EDTA (free acid): Titrated with 5 M NaOH to pH 8.0.
4.
3 M Sodium acetate solution: Dissolve 40.8 g sodium acetate (C2H3NaO2 · 3H2O) in water to a final volume of 100 mL, and adjust to pH 5.2 with glacial acetic acid.
2.2 Oligonucleotide Labeling
Author Manuscript
1.
ATP, [γ-32P]: 6000 Ci/mmol, 10 µCi/µL, 250 µCi, stored at −20 °C.
2.
T4 Polynucleotide Kinase: 10 U/µL, store at −20 °C.
3.
10 % Trichloroacetic acid (TCA): 10 g TCA dissolved to 100 mL in H2O (see Note 1).
4.
Silanized glass wool.
5.
Culture tubes: Sterile, 17 × 100 mm sterile culture tubes.
6.
Bio-Gel P-6 Fine Resin: Suspend 10 g of resin (Bio-Rad Laboratories, Hercules, CA, USA) in 250 mL of TE (pH 7.5) and autoclave for 45 min. Cool to room temperature, and then wash the resin with fivefold resin volumes TE three times. Remove excess liquid to give 1:1 slurry.
2.3 Cell Culture and Nuclear Protein Preparation
Author Manuscript
1.
Human embryonic kidney 293 cells (HEK293).
2.
Cell culture flasks: 182 cm2.
3.
Dulbecco’s modification of Eagle’s medium (DMEM).
1Water is >18 MΩ from a Millipore Synergy UV water purification unit.
Methods Mol Biol. Author manuscript; available in PMC 2016 May 31.
Jia et al.
Page 3
Author Manuscript Author Manuscript
4.
Adult bovine serum: The serum is inactivated by incubation at 56 °C for 30 min and stored at 4 °C prior to use.
5.
10× PBS: 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, and 2.4 g KH2PO4 made up to 1 L with H2O and autoclaved prior to use.
6.
1 M Dithiothreitol (DTT): Dissolve 15.4 g DTT in 100 mL of water. Aliquot and store at −20 °C for up to 6 months.
7.
1× Trypsin-EDTA.
8.
Phenylmethylsulfonyl fluoride (PMSF, 0.2 M): Dissolve 3.48 g PMFS in 100 mL of anhydrous isopropanol as a 0.2 M stock. Stored at −20 °C for up to 1 year.
9.
Nuclear extract hypotonic buffer: 10 mM HEPES, pH 7.9 at 4 °C, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM EDTA, 0.5 mM DTT (added prior to use), and 0.2 mM PMSF (added prior to use).
10. Nuclear extract low-salt buffer: 20 mM HEPES, pH 7.9 at 4 °C, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA, 25 % glycerol, 0.5 mM DTT, and 0.2 mM PMSF (both added prior to use). 11. Nuclear extract high-salt buffer: 20 mM HEPES, pH 7.9 at 4 °C, 1.5 mM MgCl2, 1.6 M KCl, 0.2 mM EDTA, 25 % glycerol, 0.5 mM DTT, and 0.2 mM PMSF (both added prior to use). 12. Nuclear extract dialysis buffer: 20 mM HEPES, pH 7.9 at 4 °C, 100 mM KCl, 20 % glycerol, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF (both added prior to use).
Author Manuscript
2.4 Electroblotting and SWB assay 1.
Electroblotting buffer: 20 % (v/v) methanol, 25 mM Tris-base, 192 mM glycine.
2.
SWB buffer: 10 mM HEPES (pH 7.9 at 4 °C), 50 mM NaCl, 10 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 50 µM ZnSO4, and 0.1 % (v/v) Tween-20.
3.
SWB blocking buffer: 5 % of nonfat dry milk in SWB buffer.
4.
6 M Guanidine hydrochloride: Dissolve 57.3 g guanidine hydrochloride (for molecular biology, ≥99 %) in 50 mL of SWB buffer, and then adjust to total 100 mL, freshly prepared prior to use.
2.5 1-Dimensional Gel Electrophoresis SWB (1DGE-SWB)
Author Manuscript
1.
30 % Acrylamide/Bis: Dissolve 29.2 g acrylamide and 0.8 g N,N′-methylene-bisacrylamide (Bis) to a final volume of 100 mL H2O.
2.
0.5 M Tris–HCl, pH 6.8: Dissolve 60.6 g Tris base in 800 mL H2O, titrate to pH 6.8 with 1 M HCl, and adjust to 1 L with H2O.
3.
1.5 M Tris–HCl, pH 8.8: Dissolve 181.7 g Tris base in 800 mL H2O, titrate to pH 8.8 with concentrated HCl, and adjust to 1 L with H2O.
Methods Mol Biol. Author manuscript; available in PMC 2016 May 31.
Jia et al.
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Author Manuscript
4.
50 % Glycerol, 0.01 % bromophenol blue (BPB): To 50 mL glycerol, add 10 mg BPB, and adjust to 100 mL with H2O.
5.
1 M Tris, pH 7.5: Dissolve 121.1 g Tris base in 800 mL H2O. Titrate to pH 7.5 with concentrated HCl. Adjust volume to 1 L with H2O and autoclave for 45 min.
6.
10 % (w/v) Ammonium persulfate (APS): Dissolve 100 mg APS (Sigma) in 1 mL H2O, prepared prior to use.
7.
β-Mercaptoethanol.
8.
Tetramethylethylenediamine (TEMED).
9.
10 % (w/v) Sodium dodecylsulfate (SDS): Dissolve 10 g of SDS in H2O to a final 100 mL; store at room temperature.
Author Manuscript
10. 5× Laemmli sample buffer: Mix 0.12 mL #2: 0.5 mL #4: 0.05 mL #7:0.2 mL # 9 on the day of use. 11. 10× Running buffer: 250 mM Tris base, 1.92 M glycine. Working solution is prepared with 30 mL 10× and 3 mL 10 % SDS and adjusted to 300 mL with H2O and should be pH 8.3. 2.6 2-Dimensional Gel Electrophoresis SWB (2DGE-SWB)
Author Manuscript
1.
Immobilized pH gradient (IPG) strip: (Bio-Rad) Ready@Strip™ IPG strips, linear 7 cm, pH 3–10.
2.
Rehydration buffer: 7 M Urea, 2 M thiourea, 2 % CHAPS, 65 mM DTT, 0.2 % BioLyte 3/10 Ampholytes (Bio-Rad), 1 % Zwittergent 3–10 (Sigma), and 0.001 % bromophenol blue.
3.
Equilibration buffer (EB): 50 mM Tris, pH 8.8, 6 M urea, 2 % (w/v) SDS, 30 % (v/v) glycerol, and 0.001 % (w/v) bromophenol blue.
4.
Reduction buffer: 2 % (w/v) DTT in EB.
5.
Alkylation buffer: 2.5 % (w/v) Iodoacetamide in EB.
2.7 EMSA-Based SWB (3DGE-SWB)
Author Manuscript
1.
5× EMSA buffer: 100 mM HEPES (pH 7.9), 0.5 mM EDTA, 250 mM NaCl, 25 mM MgCl2, 5 mM DTT, 50 % (v/v) glycerol, and 0.5 % (v/v) Tween-20. Store at −20 °C for 1 month.
2.
Poly (deoxyinosinic-deoxycytidylic) acid (poly-dI:dC): (Sigma) Dissolved in TE to bring to 0.5 mg/mL stock. Stored at −20 °C for at least 3 months.
3.
5× Loading buffer: 50 % (v/v) glycerol, 0.01 % (w/v) bromophenol blue.
4.
5× TBE buffer: 54.5 g Tris base, 27.8 g boric acid, 3.7 g Na4EDTA, pH 8.3 at room temperature.
5.
Extraction buffer: 50 mM Tris, pH 9.0, 50 mM DTT, and 0.5 % (v/v) Tween-20.
Methods Mol Biol. Author manuscript; available in PMC 2016 May 31.
Jia et al.
Page 5
Author Manuscript
3 Methods 3.1 Oligonucleotide Preparation
Author Manuscript
1.
Dissolve received oligonucleotide in 300 µL of TE (pH 7.5) buffer (see Note 2).
2.
Add 30 µL of sodium acetate solution (3 M) and 1 mL of 100 % ethanol (absolute), mix, and allow oligonucleotides to precipitate at −85 °C for 1 h (see Note 3).
3.
Collect oligonucleotides by centrifugation at 14,000 × g using bench centrifuge for 15 min at 4 °C (see Note 4).
4.
Carefully remove the supernatant and wash the pellet with 500 µL of ice-cold 70 % (v/v) ethanol by vortex mixing.
5.
Again, centrifuge and remove the supernatant as in step 4.
6.
Air-dry the oligonucleotide by leaving the tube uncapped and covered with Kimwipes for 2 h at room temperature.
7.
Dissolve the pellet in 500 µL of TE (pH 7.5) at room temperature for 30 min with occasional vortex mixing.
8.
Determine the concentration by measuring the absorption at 260 nm using 1 µL oligonucleotide diluted to 1 mL water (see Note 5).
9.
Adjust the concentration to 0.1 mM with TE (pH 7.5).
3.2 Oligonucleotide Labeling
Author Manuscript
1.
Caution: All steps must be carried out in a radioactivity control area.
2.
Mix 2 µL of 10 µM oligonucleotide, 5 µL of polynucleotide kinase buffer (10×, supplied with enzyme), 2 µL of γ-32P-ATP (10–20 µCi), and 2 µL of T4 Polynucleotide Kinase (10 U/µL), and bring to 50 µL with water in a 1.5 mL Eppendorf tube. Mix by gentle tapping; centrifuge briefly.
3.
Incubate at 37 °C for 60 min.
2Oligonucleotides are purchased from Integrated DNA Technologies (Coralville, IA, USA). Molar absorptivity (E 260nm) is provided to calculate concentration. 3For short oligonucleotides (
Methods Mol Biol. Author manuscript; available in PMC 2016 May 31. Published in final edited form as: Methods Mol Biol. 2015 ; 1334: 85–99. doi:10.1007/978-1-4939-2877-4_5.
Southwestern Blotting Assay Yinshan Jia, Linda Nagore, and Harry Jarrett
Abstract
Author Manuscript
Southwestern blotting is a technique used to study DNA-protein interactions. This method detects specific DNA-binding proteins by incubating radiolabeled DNA with a gel blot, washing, and visualizing through autoradiography. A blot resulting from 1-dimensional SDS-PAGE reveals the molecular weight of the binding proteins. To increase separation and determine isoelectric point a 2-dimensional gel can be blotted. Additional dimensions of electrophoresis, such as a gel shift (EMSA), can precede isoelectric focusing and SDS-PAGE to further improve separation. Combined with other techniques, such as mass spectrometry, the DNA-binding protein can be identified.
Keywords Transcription; Regulation; DNA; Response elements; Oligonucleotide; Promoter; Immunoblots; Nitrocellulose membrane; PVDF membrane; Southwestern blotting
Author Manuscript
1 Introduction Regulation of gene expression is essential in human development as well as pathogenesis [1]. The gene regulatory mechanism involves distinct 6–8 bp DNA motifs referred to as response elements, which bind transcription factors [2]. Transcription factors are DNAbinding proteins that interact with unique response elements at the promoter region of DNA, or other functional cisacting response elements, resulting in either gene expression or repression [1]. Identification of transcription factors specific to a particular gene is not only significant to gene regulation but also to understanding gene function. Southwestern blotting assay (SWB) is one of the most powerful techniques to explore protein-DNA interaction and transcription factor regulation.
Author Manuscript
SWB, similar to other blotting techniques, separates proteins (or DNA) by gel electrophoresis. The gel containing the separated proteins is electro-transferred (blotted) to a membrane, such as nitrocellulose (NC) or polyvinylidine difluoride (PVDF). To detect the DNA-binding proteins, the proteins are partially renatured and bound to nanomolar concentrations of radiolabeled DNA. Any unbound DNA is washed away, and then bands on the blot are detected by autoradiography. This technique is especially useful in the identification of transcription factor as it gives information on the molecular weights of all DNA-binding proteins involved with a particular sequence of DNA. An experimental procedure is shown schematically in Fig. 1.
Jia et al.
Page 2
Author Manuscript
Since the original SWB assay, first described in 1980 [3], many extensions of the SWB method have been developed [4, 5]. In this chapter, three detailed SWB methods are described that differ in the number of electrophoresis dimensions used for protein separation. 1D-SWB uses separation on a single SDS-PAGE dimension, 2D-SWB uses separation by isoelectric focusing followed by SDS-PAGE in the second dimension, while 3D-SWB uses separation by DNA gel shift on a non-denaturing gel before 2D-SWB. Coupled with proteomic techniques, several transcription factors have been identified [4]. Additionally, a method has been developed that allows multiple reprobes with different oligonucleotides on one blot [6]. Each of these SWB techniques provides alternatives to investigate regulatory transcription factors in vitro.
2 Materials Author Manuscript
2.1 Oligonucleotide Preparation 1.
Oligonucleotide: Approximately 20–25 nt in size along with its complementary (antisense) strand, containing one protein-binding motif (0.1 µmol).
2.
TE buffer: 10 mM Tris (free base), titrated to pH 7.5 with HCl, 1 mM EDTA.
3.
0.5 M EDTA (free acid): Titrated with 5 M NaOH to pH 8.0.
4.
3 M Sodium acetate solution: Dissolve 40.8 g sodium acetate (C2H3NaO2 · 3H2O) in water to a final volume of 100 mL, and adjust to pH 5.2 with glacial acetic acid.
2.2 Oligonucleotide Labeling
Author Manuscript
1.
ATP, [γ-32P]: 6000 Ci/mmol, 10 µCi/µL, 250 µCi, stored at −20 °C.
2.
T4 Polynucleotide Kinase: 10 U/µL, store at −20 °C.
3.
10 % Trichloroacetic acid (TCA): 10 g TCA dissolved to 100 mL in H2O (see Note 1).
4.
Silanized glass wool.
5.
Culture tubes: Sterile, 17 × 100 mm sterile culture tubes.
6.
Bio-Gel P-6 Fine Resin: Suspend 10 g of resin (Bio-Rad Laboratories, Hercules, CA, USA) in 250 mL of TE (pH 7.5) and autoclave for 45 min. Cool to room temperature, and then wash the resin with fivefold resin volumes TE three times. Remove excess liquid to give 1:1 slurry.
2.3 Cell Culture and Nuclear Protein Preparation
Author Manuscript
1.
Human embryonic kidney 293 cells (HEK293).
2.
Cell culture flasks: 182 cm2.
3.
Dulbecco’s modification of Eagle’s medium (DMEM).
1Water is >18 MΩ from a Millipore Synergy UV water purification unit.
Methods Mol Biol. Author manuscript; available in PMC 2016 May 31.
Jia et al.
Page 3
Author Manuscript Author Manuscript
4.
Adult bovine serum: The serum is inactivated by incubation at 56 °C for 30 min and stored at 4 °C prior to use.
5.
10× PBS: 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, and 2.4 g KH2PO4 made up to 1 L with H2O and autoclaved prior to use.
6.
1 M Dithiothreitol (DTT): Dissolve 15.4 g DTT in 100 mL of water. Aliquot and store at −20 °C for up to 6 months.
7.
1× Trypsin-EDTA.
8.
Phenylmethylsulfonyl fluoride (PMSF, 0.2 M): Dissolve 3.48 g PMFS in 100 mL of anhydrous isopropanol as a 0.2 M stock. Stored at −20 °C for up to 1 year.
9.
Nuclear extract hypotonic buffer: 10 mM HEPES, pH 7.9 at 4 °C, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM EDTA, 0.5 mM DTT (added prior to use), and 0.2 mM PMSF (added prior to use).
10. Nuclear extract low-salt buffer: 20 mM HEPES, pH 7.9 at 4 °C, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA, 25 % glycerol, 0.5 mM DTT, and 0.2 mM PMSF (both added prior to use). 11. Nuclear extract high-salt buffer: 20 mM HEPES, pH 7.9 at 4 °C, 1.5 mM MgCl2, 1.6 M KCl, 0.2 mM EDTA, 25 % glycerol, 0.5 mM DTT, and 0.2 mM PMSF (both added prior to use). 12. Nuclear extract dialysis buffer: 20 mM HEPES, pH 7.9 at 4 °C, 100 mM KCl, 20 % glycerol, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF (both added prior to use).
Author Manuscript
2.4 Electroblotting and SWB assay 1.
Electroblotting buffer: 20 % (v/v) methanol, 25 mM Tris-base, 192 mM glycine.
2.
SWB buffer: 10 mM HEPES (pH 7.9 at 4 °C), 50 mM NaCl, 10 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 50 µM ZnSO4, and 0.1 % (v/v) Tween-20.
3.
SWB blocking buffer: 5 % of nonfat dry milk in SWB buffer.
4.
6 M Guanidine hydrochloride: Dissolve 57.3 g guanidine hydrochloride (for molecular biology, ≥99 %) in 50 mL of SWB buffer, and then adjust to total 100 mL, freshly prepared prior to use.
2.5 1-Dimensional Gel Electrophoresis SWB (1DGE-SWB)
Author Manuscript
1.
30 % Acrylamide/Bis: Dissolve 29.2 g acrylamide and 0.8 g N,N′-methylene-bisacrylamide (Bis) to a final volume of 100 mL H2O.
2.
0.5 M Tris–HCl, pH 6.8: Dissolve 60.6 g Tris base in 800 mL H2O, titrate to pH 6.8 with 1 M HCl, and adjust to 1 L with H2O.
3.
1.5 M Tris–HCl, pH 8.8: Dissolve 181.7 g Tris base in 800 mL H2O, titrate to pH 8.8 with concentrated HCl, and adjust to 1 L with H2O.
Methods Mol Biol. Author manuscript; available in PMC 2016 May 31.
Jia et al.
Page 4
Author Manuscript
4.
50 % Glycerol, 0.01 % bromophenol blue (BPB): To 50 mL glycerol, add 10 mg BPB, and adjust to 100 mL with H2O.
5.
1 M Tris, pH 7.5: Dissolve 121.1 g Tris base in 800 mL H2O. Titrate to pH 7.5 with concentrated HCl. Adjust volume to 1 L with H2O and autoclave for 45 min.
6.
10 % (w/v) Ammonium persulfate (APS): Dissolve 100 mg APS (Sigma) in 1 mL H2O, prepared prior to use.
7.
β-Mercaptoethanol.
8.
Tetramethylethylenediamine (TEMED).
9.
10 % (w/v) Sodium dodecylsulfate (SDS): Dissolve 10 g of SDS in H2O to a final 100 mL; store at room temperature.
Author Manuscript
10. 5× Laemmli sample buffer: Mix 0.12 mL #2: 0.5 mL #4: 0.05 mL #7:0.2 mL # 9 on the day of use. 11. 10× Running buffer: 250 mM Tris base, 1.92 M glycine. Working solution is prepared with 30 mL 10× and 3 mL 10 % SDS and adjusted to 300 mL with H2O and should be pH 8.3. 2.6 2-Dimensional Gel Electrophoresis SWB (2DGE-SWB)
Author Manuscript
1.
Immobilized pH gradient (IPG) strip: (Bio-Rad) Ready@Strip™ IPG strips, linear 7 cm, pH 3–10.
2.
Rehydration buffer: 7 M Urea, 2 M thiourea, 2 % CHAPS, 65 mM DTT, 0.2 % BioLyte 3/10 Ampholytes (Bio-Rad), 1 % Zwittergent 3–10 (Sigma), and 0.001 % bromophenol blue.
3.
Equilibration buffer (EB): 50 mM Tris, pH 8.8, 6 M urea, 2 % (w/v) SDS, 30 % (v/v) glycerol, and 0.001 % (w/v) bromophenol blue.
4.
Reduction buffer: 2 % (w/v) DTT in EB.
5.
Alkylation buffer: 2.5 % (w/v) Iodoacetamide in EB.
2.7 EMSA-Based SWB (3DGE-SWB)
Author Manuscript
1.
5× EMSA buffer: 100 mM HEPES (pH 7.9), 0.5 mM EDTA, 250 mM NaCl, 25 mM MgCl2, 5 mM DTT, 50 % (v/v) glycerol, and 0.5 % (v/v) Tween-20. Store at −20 °C for 1 month.
2.
Poly (deoxyinosinic-deoxycytidylic) acid (poly-dI:dC): (Sigma) Dissolved in TE to bring to 0.5 mg/mL stock. Stored at −20 °C for at least 3 months.
3.
5× Loading buffer: 50 % (v/v) glycerol, 0.01 % (w/v) bromophenol blue.
4.
5× TBE buffer: 54.5 g Tris base, 27.8 g boric acid, 3.7 g Na4EDTA, pH 8.3 at room temperature.
5.
Extraction buffer: 50 mM Tris, pH 9.0, 50 mM DTT, and 0.5 % (v/v) Tween-20.
Methods Mol Biol. Author manuscript; available in PMC 2016 May 31.
Jia et al.
Page 5
Author Manuscript
3 Methods 3.1 Oligonucleotide Preparation
Author Manuscript
1.
Dissolve received oligonucleotide in 300 µL of TE (pH 7.5) buffer (see Note 2).
2.
Add 30 µL of sodium acetate solution (3 M) and 1 mL of 100 % ethanol (absolute), mix, and allow oligonucleotides to precipitate at −85 °C for 1 h (see Note 3).
3.
Collect oligonucleotides by centrifugation at 14,000 × g using bench centrifuge for 15 min at 4 °C (see Note 4).
4.
Carefully remove the supernatant and wash the pellet with 500 µL of ice-cold 70 % (v/v) ethanol by vortex mixing.
5.
Again, centrifuge and remove the supernatant as in step 4.
6.
Air-dry the oligonucleotide by leaving the tube uncapped and covered with Kimwipes for 2 h at room temperature.
7.
Dissolve the pellet in 500 µL of TE (pH 7.5) at room temperature for 30 min with occasional vortex mixing.
8.
Determine the concentration by measuring the absorption at 260 nm using 1 µL oligonucleotide diluted to 1 mL water (see Note 5).
9.
Adjust the concentration to 0.1 mM with TE (pH 7.5).
3.2 Oligonucleotide Labeling
Author Manuscript
1.
Caution: All steps must be carried out in a radioactivity control area.
2.
Mix 2 µL of 10 µM oligonucleotide, 5 µL of polynucleotide kinase buffer (10×, supplied with enzyme), 2 µL of γ-32P-ATP (10–20 µCi), and 2 µL of T4 Polynucleotide Kinase (10 U/µL), and bring to 50 µL with water in a 1.5 mL Eppendorf tube. Mix by gentle tapping; centrifuge briefly.
3.
Incubate at 37 °C for 60 min.
2Oligonucleotides are purchased from Integrated DNA Technologies (Coralville, IA, USA). Molar absorptivity (E 260nm) is provided to calculate concentration. 3For short oligonucleotides (
