EMSA gel - dirty bands! (Jul/05/2005 )
Hi
I was wondering if anyone knows how to get rid of 'gritty' appearance of EMSA gels?
When i first did it, was no problem, nice, clean lanes. But for some reason I am now getting a very gritty, dotty EMSA image when i visualise the gel. I use 32-p labelled probes (HIF-1 assay from hypoxic cells). I let the binding reaction incubate at RT for approx 25 mins then run it on 6% gel:
Large 6% poly acrylamide gel (50ml)
38 mL ml H20
1.25 mL ml 10X TBE
10 mL acrylamide 30%
700 uL APS 10%
70 uL TEMED
Please can anyone offer some help? I have made fresh buffers, fresh probes and fresh extracts. No joy
I was wondering if anyone knows how to get rid of 'gritty' appearance of EMSA gels?
When i first did it, was no problem, nice, clean lanes. But for some reason I am now getting a very gritty, dotty EMSA image when i visualise the gel. I use 32-p labelled probes (HIF-1 assay from hypoxic cells). I let the binding reaction incubate at RT for approx 25 mins then run it on 6% gel:
Large 6% poly acrylamide gel (50ml)
38 mL ml H20
1.25 mL ml 10X TBE
10 mL acrylamide 30%
700 uL APS 10%
70 uL TEMED
Please can anyone offer some help? I have made fresh buffers, fresh probes and fresh extracts. No joy
I had the same problem while back. Do you use a screen in your cassette when you expose your gel? An old screen or cracked screen will give you this result. The screen also sharpens your bands on the film
Yes the screen is cracked in my hyperfilm casette! I hadn't thought of that at all. But it was the sole reason in your case? I'll give it a try asap.
The other problem I'm having is getting a supershift to work. When i expose the gel, the supershift band is a lot more dense, but it doesnt supershift. I had just come across someone mentioning that DTT may be doing something adverse to the antibody? I am using anti-HIF alpha
Thank you so much for you suggestion
The other problem I'm having is getting a supershift to work. When i expose the gel, the supershift band is a lot more dense, but it doesnt supershift. I had just come across someone mentioning that DTT may be doing something adverse to the antibody? I am using anti-HIF alpha
Thank you so much for you suggestion
Well, how do you mix the Ab with your nuclear extract? If you mix the NE with your probe first, then add the Ab but see no supershift, the Ab may recognize the DNA binding domain of HIF. Add the Ab directly to the NE, incubate on ice 10-20' and then add probe. This gives a chance for the ab to bind before the probe gets there.
If you have speckles it may be your screen as suggested.
Most likely it is due to unincorporated nucleotides. I suggest to reanneal your
oligos, make a new probe and repurify on a column.
Also, when drying your gel, put two whatmans under the gel and make sure it is really dry.
Some people claim that putting a whatman on top of the gel also helps reduce fuzziness.
May be true, but impt. to completely dry your gel.
Have others gotten this antibody to work for supershifts?
It is possible that you need to use more antibody or use it in a more
concentrated form.
As suggested, it usually works best when added to the protein prior to addition of the probe.
Well, how do you mix the Ab with your nuclear extract? If you mix the NE with your probe first, then add the Ab but see no supershift, the Ab may recognize the DNA binding domain of HIF. Add the Ab directly to the NE, incubate on ice 10-20' and then add probe. This gives a chance for the ab to bind before the probe gets there.
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I had tried both adding before and after labelled probe addition. In both cases, the band on the gel gets a lot darker, but no supershift. I'll try doing the ab pre-incuabtion on ice this time and see what happens.
Thanks for your help!
P.s would you know of any quick protocol i could use to get rid of unincorporated nucleotides from the labelled probe, without having to use kits?
[/quote]
I had tried both adding before and after labelled probe addition. In both cases, the band on the gel gets a lot darker, but no supershift. I'll try doing the ab pre-incuabtion on ice this time and see what happens.
Thanks for your help!
P.s would you know of any quick protocol i could use to get rid of unincorporated nucleotides from the labelled probe, without having to use kits?
[/quote]
Purify by G-25 or G-50 spin column. This gets rid of all unincorporated nucleotides. Very easy to prepare the column.
Take a 1ml syringe (without needle
_) fill with 50% G-50 slurry.Spin down 3 min, 1500 rpm
Fill syringe again, spin down
add 100ul of water or TE to equibrilate - spin down.
add your probe (100 ul) and spin down
collect flow through
Nucleotides should remain in the column (check count)
Hope this helps
EDIT:
forgot to mention: MUST plug the bottom of the syringe with glass wool so beads dont go through
Hi
I was wondering did you get your supershift to work? I've been having similar problems with mine - there is a darker band in my supershift lane when I add the antibody first, but no shift. When I add the oligo first the band is not darker but similar to my nuclear extract plus oligo only control and still no shift!
I'd like to know if you managed to sort out the problem
Thanks