McClean:EMSA Protocol

Overview

This is a protocol for the LightShift Chemiluminescent EMSA Kit from Thermo Scientific (cat # 20148).

It is highly recommended to read the instructions that come with the kit. They have a lot of good information that will help with decision making.

Materials

• LightShift EMSA Optimization and Control Kit (20148X)
• Chemiluminescent Nucleic Acid Detection Module (89880)
• Biotin 3' or 5' end-labeled DNA target
• Unlabeled DNA target for competitive control.
• Positively charged nylon membrane (77016)
• 5X TBE (450mM Tris, 450mM boric acid, 10mM EDTA, pH8.3) (LC6675)
• CCD camera
• UV lamp or crosslinking equipment with 254nm bulbs or 312nm transilluminator
• Electrophoresis apparatus
• Electroblotter or capillary transfer apparatus with needed buffer
• High-quality blotting paper (88600)
• Membrane forceps
• Polyacrylamide gel in 0.5X TBE (EC63655BOX)

Protocol

A. Plan the Binding Reactions

Binding Reactions for Control EBNA System

Component	Final Concentration	Reactions
	Reaction #	#1(ul)	#2(ul)	#3(ul)
Ultrapure Water	----	12	11	9
10X Binding Buffer	1X	2	2	2
50% Glycerol	2.5%	1	1	1
100mM MgCl2	5mM	1	1	1
1ug/ul Poly(dIdC)	50ng/ul	1	1	1
1% NP-40	0.05%	1	1	1
Unlabeled EBNA DNA	4pmol	----	----	2
EBNA Extract	1 unit	----	1	1
Biotin-EBNA Control DNA	20fmol	2	2	2
Total Volume of Reaction	----	20	20	20

Binding Reactions for Experimental Samples

Component	Final Concentration	Reactions
	Reaction #	#4(ul)	#5(ul)	#6(ul)
Ultrapure Water	----	12	11	9
10X Binding Buffer	1X	2	2	2
Optional: 50% Glycerol	2.5%	1	1	1
Optional: 100mM MgCl2	5mM	1	1	1
Optional: 1ug/ul Poly(dIdC)	50ng/ul	1	1	1
Optional: 1% NP-40	0.05%	1	1	1
Unlabeled Target DNA	4pmol	----	----	2
Protein Extract (2-3ul)	----	----	1	1
Biotin End-Labeled Target DNA	20fmol	2	2	2
Total Volume of Reaction	----	20	20	20

• Notes: The amount of protein extract used is dependent on the system.

• You need to experiment with various salts and detergents to determine conditions and amounts for binding.

• Other options: You may want to add KCl and/or ZnCl and/or EDTA to the reactions depending on your protein.

• If you are looking at a zinc-finger protein, be careful with your EDTA as it will chelate the zinc.

B. Prepare and Pre-run Gel

1. Use a pre-cast 6% DNA retardation gel in 0.5X TBE. (cat # EC63655BOX )
2. Place the gel in the electrophoresis unit, and clamp it to obtain a seal. Fill the inner chamber with 0.5X TBE to a height several 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-electrophorese the gel for 30-60 minutes. Apply 100V for an 8 × 8 × 0.1cm gel.
3. Proceed to Section C while gel is pre-electrophoresing.

C. Prepare and Perform Binding Reactions

1. Thaw all binding reaction components and place them on ice.
2. Prepare complete sets of 20ul binding reactions according to Procedure Section A; 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 several times to mix. DO NOT vortex or mix vigorously.

D. Electrophorese Binding Reactions

1. Switch off current to the electrophoresis gel.
2. Flush the wells and then load 15μL of each sample onto the polyacrylamide gel.
3. Switch on current (set to 100V for 8 × 8 × 0.1cm gel) and electrophorese samples until the bromophenol blue dye has migrated approximately 2/3 to 3/4 down the length of the gel.

E. Electrophoretic Transfer of Binding Reactions to Nylon Membrane

• Note: Use clean transfer sponges. Avoid using sponges that have been used in Western blots.

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. Use 0.5X TBE cooled to ~10ºC with a circulating water bath. Use very clean forceps and powder-free gloves, and handle the membrane only at the corners.
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

1. Crosslink for 10-15 minutes with the membrane face down on a transilluminator equipped with 312nm bulbs.
2. After the membrane is crosslinked, proceed directly to Section G. Alternatively, the membrane may be stored dry at room temperature for several days. Do not allow the membrane to get wet again until ready to proceed with Section G.

G. Detect Biotin-labeled DNA by Chemiluminescence

• The recommended volumes are for an 8 × 10cm membrane. If larger gels are used, adjust volumes in Section G accordingly. Perform all blocking and detection incubations in clean trays or in plastic weigh boats on an orbital shaker.
• Note: The conjugate/blocking buffer solution has been optimized for the Nucleic Acid Detection Module and should not be modified.
• Note: Exposure to the sun or any intense light can harm the Working Solution. Keep the Working Solution in an amber bottle and avoid prolonged exposure to intense light. Short-term exposure to typical laboratory lighting will not harm the Working Solution.

1. Gently warm the Blocking Buffer and the 4X Wash Buffer to 37-50°C in a water bath until all particulate is dissolved. These buffers may be used between room temperature and 50°C as long as all particulate remains in solution. The Substrate Equilibration Buffer may be used between 4°C and room temperature.
2. To block the membrane add 20mL of Blocking Buffer and incubate for 15 minutes with gentle shaking.
3. Prepare conjugate/blocking buffer solution by adding 66.7μL Stabilized Streptavidin-Horseradish Peroxidase Conjugate to 20mL Blocking Buffer (1:300 dilution).
4. Decant blocking buffer from the membrane and replace it with the conjugate/blocking solution. Incubate membrane in the conjugate/blocking buffer solution for 15 minutes with gentle shaking.
5. Prepare 1X wash solution by adding 40mL of 4X Wash Buffer to 120mL of ultrapure water.
6. Transfer membrane to a new container and rinse it briefly with 20mL of 1X wash solution.
7. Wash membrane four times for 5 minutes each in 20mL of 1X wash solution with gentle shaking.
8. Transfer membrane to a new container and add 30mL of Substrate Equilibration Buffer. Incubate membrane for 5 minutes with gentle shaking.
9. Prepare Substrate Working Solution by adding 5mL Luminol/Enhancer Solution to 5mL Stable Peroxide Solution.
10. Remove membrane from the Substrate Equilibration Buffer, carefully blotting an edge of the membrane on a paper towel to remove excess buffer. Place membrane in a clean container or onto a clean sheet of plastic wrap placed on a flat surface.
11. Pour the Substrate Working Solution onto the membrane so that it completely covers the surface. Alternatively, the membrane may be placed DNA side down onto a puddle of the Working Solution. Incubate membrane in the substrate solution for 5 minutes without shaking.
12. Remove membrane from the Working Solution and blot an edge of the membrane on a paper towel for 2-5 seconds to remove excess buffer. Do not allow the membrane to become dry.
13. Expose membrane to an appropriately equipped CCD camera.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. Anecdotal observations that might be of use to others can also be posted here.

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