Trouble with ugly bands in EMSA - (Mar/19/2007 )
Hi,i'm doing the gel shift research using the LIGHTSHIFT chemiluminescent EMSA kit (pierce company).
With the provided control primers and nuclear extract in kit ,I can obtain a good result (Figure1 Left).But when using the tested primers and nuclear extracts ,the results were very odd and puzzling (Figure1 right). Especially in the electrophoresis process,a very bent,blunt and ugly shifting band can be observed in the gel .(Figure 2) .The primers were designed according to the published papers. Method of Preparation of Nuclear Extracts for Gel Shifts and Westerns Blots is as follow from Dr.Mirmira website.
I have changed all the possible condition including the electrophoresis buffer (TBE ,TGE),primers ,Gel , and so on . I am frustrated by the result. Can anyone tell me the cause,Thanks in advance.
The following protocol is optimized for about 107 cells (near confluent 10 cm plate). NOTE: ALL COMPONENTS (BUFFERS, PROTEASE INHIBITORS, ETC) MUST BE KEPT ON ICE DURING THE ENTIRE PROCEDURE.
1. 1. Wash plates with PBS two times.
2. 2. Prepare 5 ml of Buffer A in a 15 ml conical centrifuge tube, and add the following:
-5 ul of each protease inhibitor (leupeptin, PMSF, aprotinin, pepstatin, and DTT)
-200 ul of 10% IGEPAL.
Add 0.5 ml of this preparation directly to each plate, and wait 10 min. at room temp.
3. 3. Scrape cells with NEW sterile scraper, then pipet up and down with P1000 several times to disrupt cell clumps. NOTE: you will have nearly 1 ml of lysate at this point.
4. 4. Transfer this lysate to a 1.5 microcentrifuge tube, place on ice.
5. 5. Centrifuge at 4 C at top speed (15000 x g) for 3 min.
6. 6. Place tubes on ice.
7. 7. Save supernatant (cytosolic fraction) for luciferase/ß-gal assay, if desired. Otherwise, discard the supernatant.
8. 8. Prepare 1 ml of Buffer B in a 1.5 ml eppendorf, and add 1 ul of each protease inhibitor as above, but this time DO NOT add IGEPAL. Add 150 l of this buffer to each tube, and resuspend pellet by pipeting up and down with a P200. Place on ice.
9. 9. Shake vigorously at 4 C for 2h.
10. 10. Centrifuge at 4 C, top speed for 5 min. Measure Bradford (protein) concentration using 5 ul of each sample, then aliquot 15 ul aliquots into prechilled 0.5 ml prelabeled microcentrifuge tubes, freeze in liquid nitrogen, and store in –80 C freezer.
10 mM HEPES, pH 7.9
10 mM KCl
0.1 mM EDTA
Add just before use: 1mM DTT, 0.5 mM PMSF, 5 ul of 10 ug/ul of aprotinin, leupeptin, and pepstatin A to 5 ml of buffer.
20 mM HEPES, pH 7.9
0.4 M NaCl
1 mM EDTA
Add just before use: DTT, PMSF, aprotinin, leupeptin, pepstatin A as above.
what is the difference in glycerol and detergent concentration between your control nuclear extracts, and the nuclear extracts you made yourself for testing?
glycerol in particular can cause problems like that in your gel, if you add way too much.