EMSA Binding Reaction Components - Troubleshooting start point - Lightshift Chemiluminescent EMSA (Pierce) (Apr/08/2008 )
Shortly ago I started working with the non-radioactive EMSA kit from Pierce. I was successful with getting the control reactions that came with the kit to produce the expected shifts but when it comes to working with my own probes, I have some questions. Namely, I've tried binding with my own probes (one of which theorectically would produce a shift for sure) but instead, all lanes that I ran had detectable but non-shifted bands.
I suspect this has to do with the binding step. Thus far I've been following values recommended by protocol: 1x binding buffer, 50 ng/ul (in 20 ul) Poly(dI-dC), 20 fmol biotin labelled probes.
1) How much protein is usually used for binding? I used about 4 ug of crude nuclear extract (isolated using the Schreiber method) I think this is more than enough.
2) The kit lists optional binding components: Glycerol, NP-40, KCl, MgCl2, EDTA > According to the protocol, these ''have effects on determining the shift'' but how? Trying all possible combinations seem a little unrealistic especially since I don't know in what amounts. Any recommendations?
3) Since this is a non-radioactive kit, probes are labelled with biotin. I used the Pierce 3' labeling kit and was successful with getting a signal when I blotted the labelled probes to membrane. My question is, will biotin labeling interfere with protein interactions with the probe and is it general practice to label both ss oligo for a given probe before annealing. At the moment, my annealing step consists of heating the probes to Tm for 30 min and letting it cool to RT but is there a way to check for sucessful annealing?
Any advice would be appreciated! THX รถ)
You don't say what is in the "binding buffer" but the usual approach is to start with conditions as similar as possible to the native protein/DNA binding conditions. This would certainly include some amount of both KCl and Mg++ (probably no EDTA). Glycerol might help with protein stability. Read about the normal cell salt concentrations of your cell strain of choice.
I would be annealing the oligos by heating to higher than Tm, probably 95C or so. 30 minutes is way too long, you need only a few seconds. You can cool them by heating a beaker of water and putting the tube into it, letting the water cool on the bench.
The biotin could interfere with binding, depending on how close the biotin is to the DNA binding site. Usually biotin is added with a long linker to minimize steric effects. Usually only one oligo need be labeled.