DRAFTMarch 21, 2007 5:46 pm
EMSA "Gel Shift" Kit
F, 2007 5:46 pmEMSATtile.fm
About the User Manual
Who Should Read this Manual
Anyone that has purchased an EMSA “Gel Shift” Kit from Panomics to measure the binding activity of a specific TF from nuclear extracts.
This manual provides the following:
♦Kit contents and storage conditions
CAUTIONAll chemicals should be considered potentially hazardous. We recommend that this product and its components be handled by those trained
in laboratory techniques and be used according to the principles of good laboratory practice.
Note This product is intended for research use only. It is not for diagnosis of disease in humans or animals.
About the EMSA “Gel
Introduction to EMSA Kits
Panomics’ Electrophoretic-Mobility Shift Assay (EMSA) Kits are useful
tools for identifying proteins that interact with DNA. This rapid technique
Panomics’ EMSA Kit Contents and Storage Conditions
Kit Contents and Storage
The EMSA Kit contains the following components. Refer to the
product insert for quantities and details of components supplied. If stored
Required Equipment and Materials Not Provided
30% Acrylamide/Bis Solution
Ammonium Persulfate (APS)
10X TBE Stock Solution (1.0 M Tris, 0.9M Boric Acid 0.01 M EDTA)
Nuclear Extraction Kit
Protein Determination Kit
UV Cross Linker (optional)
Chemiluminescent Imaging System (optional if using X-Ray film)
X-Ray Film (optional if using Chemiluminescent Imaging system)
Neutrally Charged Nylon Membrane, 0.45 μm
Guidelines for Assay Design and Analysis
Preparing SamplesProtein concentration of sample inputs should be at least 0.5–2 μg/μl.
General Guidelines♦ Read this user manual and all product inserts before performing the assay.
♦Store all reagents at the recommended temperatures.
♦Use Panomics’ Nuclear Extraction Kit for best results.
Before You Start
♦ Thaw Positive Control and sample nuclear extracts on ice.
♦Thaw TF-Specific Probe and TF-Specific Cold Probe on ice.
Preparing Nuclear Extracts
We recommend using a commercially available kit, such as Panomics’ Nuclear
Extraction Kit (P/N AY2002). Please note that the provided control nuclear extract is a
positive control only when used with the supplied control probe.
♦We recommend using a concentration of ~5–10 μg/μl of Nuclear Extract. If your
samples have a different concentration, adjust the amount of water accordingly.
♦When measuring protein concentration, we also recommend using the DC
Protein assay from Bio-Rad (Lowry assay).
Forming TF-DNA Complexes
2For each nuclear extract sample, combine the following components into a sterile 0.5-ml microcentrifuge tube (PCR tube).
• 1.0 μl Nuclear Extract
• 1.0 μl Poly d(I-C) (1μg/μL)
• 2.0 μl of 5X Binding Buffer
• 5.0 μl nuclease-free water
Mix above reagents and incubate at RT for 5 minutes
3Add 1.0 μl of TF Probe and proceed to step 6. Total volume should be 10 μl.
4(OPTIONAL) If a competition assay is desired (see Figure1, Lane 4), follow steps 4
• 1.0 μl Nuclear Extract
• 1.0 μl Poly d(I-C) (1μg/μL)
• 2.0 μl of 5X Binding Buffer
• 3.0 μl nuclease-free water
Mix and incubate 5 min at room temp
5Add 2.0 μl Cold TF Probe to the above mixture and incubate for 5 minutes at RT.
Then add 1.0 μl of labeled TF Probe and proceed to Step 6.
6Incubate samples at 15 °C for 30 min in a thermal cycler
Gel PreparationNon-Denaturing Gel
1Prepare a 6.0% non denaturing polyacrylamide gel. Be sure to dilute solutions/buffers, as described in the Appendix. Mix the following components
into a sterile 50-ml centrifuge tube (add in order listed):
• 1 ml of 10X TBE
• 4 ml of 30% Acrylamide/Bis
• 625 μL of 80% Glycerol
• 14.375 ml of deionized, sterile water
• 300 μL of 10% APS
• 20 μL Temed
Total volume is 20 mL
2Cast the gel as per standard protocol.
3Pre-chill 0.5X TBE to 4 °C before running your gel.
4Run gel in chilled 0.5X TBE for 10 min at 120V before loading samples into gel. Prior to loading your samples, flush the wells with a transfer pipet.
5Mix samples with 1 μL of Loading Dye provided and load 10 μL of sample to each lane.
6Run the gel at 4°C (in an ice bath or refrigerator) at 120V until the dye reaches 1 inch from the bottom of the gel (Approx. time: 50-55 min).
Transfer Standard “wet-transfer” electroblotting procedure
1Presoak Pall Biodyne B nylon membrane in 0.5X TBE. Prepare four sheets of gel-sized Whatman 3MM paper (8 x 10 cm). Presoak two sheets of Whatman
3MM paper in 0.5X TBE.
IMPORTANTPlease ensure that only Pall Biodyne B nylon membrane is used. Any other membrane may cause high backgrounds
2After electrophoresis, carefully remove one glass from the gel. Cover with one sheet of dry Whatman 3MM paper and the gel will stick to the paper.
Gently lift the Whatman paper and gel away from the glass plate and note the orientation of the gel. Add an additional Whatman paper and soak in
0.5X TBE. Sandwich the gel with the pre-soaked Biodyne B membrane and two sheets of presoaked Whatman paper then add the fiber pads on both
3Place the sandwich in an electoblotting device and fill tank with 0.5X TBE. Transfer for 30 minutes at 300mA.
IMPORTANTEnsure that the sandwich is oriented properly. The Biodyne B membrane should be closest to the positive pole (red) and the polyacrylamide
gel should be closest to the negative pole (black).
Immobilization and DetectionCrosslinking and Detection
1After transfer, remove the membrane from the sandwich of Whatman paper and gel and place between two fresh sheets of Whatman 3MM paper.
IMPORTANTIf transfer was successful, the loading dye should be slightly visible on the membrane.
2The oligos on the membrane can be fixed by baking the membrane for 1 hour in a dry oven at 80°C. Alternatively, the oligos can be fixed using a
UV crosslinker for 3 min. If UV crosslinking, ensure that the side of the membrane that was closest to the gel is exposed to the UV light source.
NoteAt this point in the experiment, you can choose to continue or stop at this point. The membrane is stable for several months if stored
between Whatman paper and in the dark.
3Transfer the membrane to a new container containing 20 ml of 1X Blocking Buffer. If more than 1 membrane was prepared, each membrane will need
its own container. Lids from a 200 μl pipette tip box can be used for 8 x 10 cm blots.
4Block the membrane by incubating at room temperature with the 1X Blocking Buffer for 15 min with gentle shaking.
5Remove 1 ml of the 1X Blocking Buffer from the blot container and place into a clean microcentrifuge tube. Add 20 μl of Streptavidin-HRP to the tube and
vortex for 10 seconds.
6Transfer the diluted Streptavidin-HRP mixture to the container with the blot and continue shaking at room temperature for another 15 min.
IMPORTANTWhen adding the mixture to the container, avoid pouring the contents of the microcentrifuge tube directly onto the membrane
7Decant the diluted Steptavidin-HRP solution. Wash each membrane for 8 minutes, 3 times at room temperature with 20 ml of 1X Wash Buffer.
8Add 20 ml of 1X Detection Buffer to each membrane and incubate at room temperature for 5 min.
9Take a plastic sheet protector and cut the sheet protector so that the membrane fits between the inside of the pocket of the two sheets.
Alternatively two pieces of transparency film can be cut and the membrane sandwiched between each piece.
Prepare 2 ml of
working Substrate Solution for each membrane by mixing (in order): 200 μl
Solution I with 200 μl Solution II, briefly vortex,
11On a flat and even surface, remove the top plastic sheet of the “sandwiched” membrane and pipette 2 ml of the mixed working Substrate Solution onto
each membrane. Replace the top plastic sheet and ensure that the substrate solution is evenly distributed over the membrane with no air bubbles.
Incubate at roomtemperature for 5 min.
IMPORTANTRemove excess substrate by gently applying pressure over the top sheet and using a paper towel to wipe up any excess fluid.
12Expose the membranes using either Hyperfilm ECL (2-10 minutes) or a chemiluminescent imaging system (12-15 min), such as the FluorChem Imager
from Alpha Innotech Corp.
IMPORTANTSeveral different exposure times to film or the imaging system may be needed for an optimal EMSA image.
Possible Problems and Recommended Solutions
♦100 ml or 1X Blocking Buffer: To 50 ml of deionized water, add 50 ml of 2X
Blocking Buffer. Mix well and store at 4°C
♦300ml of 1X Wash Buffer: To 270 ml of deionized water, add 30 ml of 10X Wash
Buffer. Mix Well and store at room temperature
♦100ml of 1X Detection Buffer: To 90 ml of deionized water, add 10 ml of 10X
Detection Buffer (provided). Mix well and store at room temperature.
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