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INSTRUCTIONS
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Description
20148 LightShift Chemiluminescent EMSA Kit , contains components for 100 binding reactions and sufficient detection reagents for approximately 1000cm 2 of membrane Kit Contents:
10X Binding Buffer , 1mL, 100mM Tris, 500mM KCl, 10mM DTT; pH 7.5, store at -20°C Biotin–EBNA Control DNA , 50μL , 10fmol/μL in 10mM Tris, 1mM EDTA; pH 7.5, store at -20°C
The 60 bp biotin end-labeled duplex contains the following binding site:
5' TAGCATATGCTA (3)
a level3'-…ATCGTATACGAT…-BIOTIN 5'
Unlabeled EBNA DNA , 50μL , 2pmol/μL in 10mM Tris, 1mM EDTA; pH 7.5, store at -20°C
The ~25 bp duplex contains the following binding site:
5'-...TAGCATATGCTA (3)
3'-...ATCGTATACGAT (5)
Epstein-Barr Nuclear Antigen (EBNA) Extract , 125μL , store at -20°C Poly (dI•dC), 125μL , 1µg/μL in 10mM Tris, 1mM EDTA; pH 7.5, store at -20°C 50% Glycerol , 500μL , store at -20°C 1% NP-40, 500μL , store at -20°C 1 M KCl , 1mL, store at -20°C 100mM MgCl 2, 500μL , store at -20°C 200mM EDTA pH 8.0, 500μL , store at -20°C
5X Loading Buffer , 1mL, store at -20°C
Stabilized Streptavidin-Horradish Peroxida Conjugate , 1.5mL, store at 4°C Chemiluminescent Substrate, stable for 6 months at room temperature or 1 year at 4°C Luminol/Enhancer Solution , 80mL
Stable Peroxide Solution , 80mL
Blocking Buffer , 500mL, store at 4°C 4X Wash Buffer , 500mL, store at 4°C
Substrate Equilibration Buffer , 500mL, store at room temperature or 4°C
Storage : Upon receipt store individual components as indicated above. Box 20148X is shipped with dry ice. Box 89880 is shipped with an ice pack.
LightShift ®
Chemiluminescent EMSA Kit
Table of Contents
Introduction (2)
Procedure for Electrophoretic Mobility Shift Assay (EMSA) (3)
A. Plan Binding Reactions (3)
2020全国高考语文B. Prepare and Pre-Run Gel (4)
C. Prepare and Perform Binding Reactions (5)
D. Electrophore Binding Reactions (5)
E. Electrophoretic Transfer of Binding Reactions to Nylon Membrane (5)
F. Crosslink Transferred DNA to Membrane (5)
G. Detect Biotin-labeled DNA by Chemiluminescence (6)
Additional Information Available from the Pierce Web Site (6)
Troubleshooting (7)
Related Thermo Scientific Products (7)
References (8)
Introduction
The electrophoretic mobility shift assay (EMSA) has been ud extensively for studying DNA-protein interactions.1-3 This technique is bad on the fact that DNA-protein complexes migrate slower than non-bound DNA in a native polyacrylamide or agaro gel, resulting in a “shift” in migration of the labeled DNA band.
The Thermo Scientific LightShift Chemiluminescent EMSA Kit us a nonisotopic method to detect DNA-protein interactions. Biotin end-labeled DNA containing the binding site of interest is incubated with a nuclear extract or purified factor. This reaction is then subjected to gel electrophoresis on a native polyacrylamide gel and transferred to a nylon membrane. The biotin end-labeled DNA is detected using the Streptavidin-Horradish Peroxida Conjugate and the Chemiluminescent Substrate.
Additional Materials Required
•Biotin 3' or 5' end-labeled DNA target. U existing end-biotinylated DNA targets or prepare them us
ing a biotin end-labeling kit (e Related Thermo Scientific Products). Do not u probes with internal biotin labels (i.e., targets
biotinylated at sites other than the 3' or 5' end, such results from random prime labeling methods) becau the internal labels may inhibit binding of the DNA binding protein.
•Positively charged nylon membrane (e Related Thermo Scientific Products)
•5X TBE (450mM Tris, 450mM boric acid, 10mM EDTA, pH 8.3)
•X-ray film (e Related Thermo Scientific Products) or CCD camera
•UV lamp or crosslinking device equipped with 254nm bulbs or 312nm transilluminator
•Electrophoresis apparatus
•Electroblotter or capillary transfer apparatus
•High-quality blotting paper
•Circulating water bath
•Plastic forceps
•Polyacrylamide gel in 0.5X TBE
Procedure for Electrophoretic Mobility Shift Assay (EMSA)
This kit has been optimized for u with polyacrylamide mini (8 × 8 × 0.1cm) gels. For larger gels, adjust electrophoresis conditions and detection reagent volumes accordingly.
A.Plan Binding Reactions
•Understanding the Control EBNA System
Include a complete t of three reactions each time an EMSA is performed. The reactions and expected results for the Control Epstein-Barr nuclear antigen(EBNA) System, which is included with the kit, are described in Table 1.
The Control EBNA System results reported in Table 1 were generated using binding reactions prepared according to Table 2. Each 20μL binding reaction contains 20 fmol of Biotin-EBNA Control DNA. Reactions were electrophored, transferred and detected according to the steps in Sections B-G of this protocol. If the kit is being ud for the first time, perform the Control EBNA System reactions to verify that the kit components and overall procedure are working properly.
Table 2. Binding reactions for Control EBNA System.
Component Final Amount
Control Reactions
#1 #2 #3
Ultrapure Water ---- 12μL11μL9μL 10X Binding Buffer (20148A) 1X 2μL2μL2μL 50% Glycerol (20148F) 2.5% 1μL1μL1μL 100mM MgCl2 (20148I) 5mM 1μL1μL1μL 1µg/μL Poly (dI•dC) (20148E)50 ng/µL 1μL1μL1μL 1% NP-40 (20148G) 0.05% 1μL1μL1μL Unlabeled EBNA DNA (20148C) 4 pmol ----- ----- 2μL EBNA Extract (20148D) 1 Unit ----- 1μL1μL Biotin–EBNA Control DNA (20148B) 20 fmol 2μL2μL2μL Total Volume ---- 20μL20μL20μL
•Planning and optimizing the Test System
As with the Control EBNA System, a complete t of three reactions should be performed with the Test System. U Table 3 as a guide for planning the Test System binding reactions. If specific binding conditions are not already known, u only minimal reaction components; e.g., 10X binding chine book
乱世家人buffer and Poly (dΙ•dC), together with the biotin-labeled target DNA, protein extract and unlabeled DNA of the Test System.
菲律宾严重车祸Nuclear protein extracts prepared using the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Reagents (e Related Thermo Scientific Products) are an excellent source of target protein. U 2-3μL of NE-PER®Nuclear Extract per 20μL binding reaction. If a greater volume of NE-PER Extract is required, remove excess salts in the extract by dialyzing into a buffer containing 200mM salt (u a Slide-A-Lyzer®MINI Dialysis Unit; e Related Thermo Scientific Products) before u in the LightShift EMSA Kit.
Optimization of the Test System can be achieved by adding other components supplied in the kit such as KCl,4, 5 glycerol, MgCl24, 6 and detergent 7, 8and determining their effects on the shift. Bovine rum albumin and basic peptides have also been shown to enhance some DNA-protein interactions.8-10 Too much glycerol in the binding reactions may cau vertical streaks along the edges of the lanes.
Poly (dI•dC), which is included in the kit, is the no nspecific competitor DNA of choice for most systems. However if the Test System target DNA quence is GC-rich, try Poly (dA•dT), sonicated c
alf thymus, salmon sperm or Escherichia coli DNA. The order of addition of the nuclear extract and biotin-labeled target DNA may affect the specificity of the DNA-protein complexes. Always add the binding reaction components in the order listed in Table 3. To overcome strong nonspecific interactions, a short pre-incubation may be required before adding the biotin-labeled target DNA.
Table 3. Binding reactions for the Test System.
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B.Prepare and Pre-Run Gel
wave1.Prepare a native polyacrylamide gel in 0.5X TBE or u a pre-cast DNA retardation gel. The appropriate polyacrylamide
percent depends on the size of the target DNA and the binding protein. Most systems u a 4 -6% polyacrylamide gel in
0.5X TBE.
2.Place the gel in the electrophoresis unit, and clamp it to obtain a al. Fill the inner chamber with 0.5X TBE to a height
veral millimeters above the top of the wells. Fill the outside of the tank with 0.5X TBE to just above the bottom of the wells, which reduces heat during electrophoresis. Flush wells and pre-electrophore the gel for 30-60 minutes. Apply 100V for an 8 × 8 × 0.1cm gel.
3.Proceed to Section C while gel is pre-electophoresing.
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C.Prepare and Perform Binding Reactions
Notes:
•Include controls in the assay to ensure the system is working properly (e Procedure, Section A).
•Do not vortex the Control DNA or the EBNA extract.
1.Thaw all binding reaction components, EBNA Control System components and Test System samples, and place them on
ice. Do not thaw the EBNA Extract until immediately before u. Thaw the EBNA Extract at room temperature. DO NOT heat the EBNA Extract, which includes thawing in your hand.
2.Prepare complete ts of 20 binding reactions for the Control EBNA System and/or the Test System according to
Procedure Section A, Tables 2 and 3; add the reagents in the order listed in the tables. Do not vortex tubes at any time during this procedure.
3.Incubate binding reactions at room temperature for 20 minutes.
4.Add 5µL of 5X Loading Buffer to each 20µL binding reaction, pipetting up and down veral times to mix. DO NOT
vortex or mix vigorously.
D.Electrophore Binding Reactions
1.Switch off current to the electrophoresis gel.
2.Flush the wells and then load 20μL of each sample onto the polyacrylamide gel.
3.Switch on current (t to 100V for 8 × 8 × 0.1cm gel) and electrophore samples until the bromophenol blue dye has
migrated approximately 2/3 to 3/4 down the length of the gel. The free biotin-EBNA Control DNA duplex migrates just behind the bromophenol blue in a 6% polyacrylamide gel.
E.Electrophoretic Transfer of Binding Reactions to Nylon Membrane
1.Soak nylon membrane in 0.5X TBE for at least 10 minutes.
2.Sandwich the gel, nylon membrane and blotting paper in a clean electrophoretic transfer unit according the
manufacturer’s instructions. U 0.5X TBE cooled to ~10ºC with a circulating water bath. U very clean forceps and powder-free gloves, and handle the membrane only at the corners.
Note: U clean transfer sponges. Avoid using sponges that have been ud in Western blots.
3.Transfer at 380mA (~100V) for 30 minutes. Typical transfer times are 30-60 minutes at 380mA using a standard tank
transfer apparatus for mini gels (8 × 8 × 0.1cm).
4.When the transfer is complete, place the membrane with the bromophenol blue side up on a dry paper towel. (There
should be no dye remaining in the gel.) Allow buffer on the membrane surface to absorb into the membrane. This will only take a minute. Do not let the membrane dry. Immediately proceed to Section F.
F.Crosslink Transferred DNA to Membrane
Three options are available for crosslinking:
•Option 1: Crosslink at 120mJ/cm2using a commercial UV-light crosslinking instrument equipped with 254nm bulbs (45-60 cond exposure using the auto crosslink function).
•Option 2: Crosslink at a distance of approximately 0.5 cm from the membrane for 5-10 minutes with a hand-held UV lamp equipped with 254nm bulbs.
•Option 3: Crosslink for 10-15 minutes with the membrane face down on a transilluminator equipped with 312nm bulbs. After the membrane is crosslinked, proceed directly to Section G. Alternatively, the membrane may be stored dry at room temperature for veral days. Do not allow the membrane to get wet again until ready to proceed with Section G.
