EMSA help...please URGENT

[sumo1]

Hello all,

a newcomer to this forum. Right now am in a mess....EMSA mess

All of last year i was doing EMSA and getting a good shift. I was using IVT/T protein in wheat germ extract. I was using empty wheat germ extract as a negative control. What other control/s do i need???

The supershift is not working at all....tried numerous conditions.

Has anyone done EMSA by overexpressing a tag(flag)-protein in say..HEK293 cells and made a nuclear extract from the transfected cells an then do a super shift with anti-flag?

do reply

K

[sumo1]

Hell again, actually i forgot to mention one thing....

i used to use empty wheat germ extract as a negative control.....now (rather too late) it struck me that i should have some thing IVT/Ted in the wheat germ with the parental vector as my proteins...if my proteiin is in pCDNA3-XYZ...i should have negative wheat germ IVT/Ted a basic empty pCDNA3 vector, right??

the sadddddest pat is that the vector alone also shows a shift which is same as the shift of my protein. What would a empty vector make and that binds to the DNA probe???? its basically empty...just the multiple coning sites are there after the SP6 promoter region.

this thing is driving me NUTS!!! so does this mean that all these days the shift i thot was actually some thing non-specific????

thats not fair!!!

[admin]

Hey Sumo! welcome to the forum.

the negative control having a shift is not good, but you may have mixed samples.

what i would do is for supershifting, make sure u use very little antibody.

for controls, also do competitions with specific (cold) and non-specific competitors (cold ie poly-dC).

[sumo1]

Originally Posted by admin

Hey Sumo! welcome to the forum.

the negative control having a shift is not good, but you may have mixed samples.
what i would do is for supershifting, make sure u use very little antibody.

for controls, also do competitions with specific (cold) and non-specific competitors (cold ie poly-dC).

Well Admin...i wish that was true...but no mixing happened.


I use 1ug of anti-FLAG..and i tried with and without DTT..still no shift...i am thinking n ow..maybe becoz the protein was not binding to the probe in the first place...because the shift was actually from the vector

yes, i did S and NS cold compete...but u see since the vector was binding the probe...it was competed and not competed out respectively with the above mentioned cold competitors.

I still wonder..what could have been IVT/Ted from a empty vector that binds so well to our probe.

is there any other way that i can prove that the shift is from my protein and not the vector??
Ours is a big protein ~100kDa...do u think it will be worthwhile trying in 59:1 bis-acryl ratio...basically trying bigger pore size?? I see some of the probe sticking in the wells...do u think that the protein-DNA complex is not able to come out of the well since the pore size is small (29:1..i use )??

I saw canalban's emsa..pain pics...they were so beautiful...wish mine were too

thanks for replying admin

K

[admin]

Hey Sumo,

IVT/T in vitro translation ie Rabbit reticulocyte lysate has many proteins that could potentially bind to your probe... unless you purified it after IVT/T well....

i usually run 6%-10% gels... i do denaturing and non-denaturing EMSAs

denaturing EMSAs are 8-10% while non-denaturing start at 6%...

not sure it would be nice to see a scan....

[Canalba]

Originally Posted by sumo1

Well Admin...i wish that was true...but no mixing happened.


I use 1ug of anti-FLAG..and i tried with and without DTT..still no shift...i am thinking n ow..maybe becoz the protein was not binding to the probe in the first place...because the shift was actually from the vector

yes, i did S and NS cold compete...but u see since the vector was binding the probe...it was competed and not competed out respectively with the above mentioned cold competitors.

I still wonder..what could have been IVT/Ted from a empty vector that binds so well to our probe.

is there any other way that i can prove that the shift is from my protein and not the vector??
Ours is a big protein ~100kDa...do u think it will be worthwhile trying in 59:1 bis-acryl ratio...basically trying bigger pore size?? I see some of the probe sticking in the wells...do u think that the protein-DNA complex is not able to come out of the well since the pore size is small (29:1..i use )??

I saw canalban's emsa..pain pics...they were so beautiful...wish mine were too

thanks for replying admin

K

Sumo1...

Thanks for the mention! I have read your post a wee while ago and have been thinking about it. I am also relatively new to the world of EMSAs but I have done a fair bit of digging round for info, plus have got loads of help from this forum - you are in the right place
Several thoughts and things you could try:

1)Could it be that your lysate concentration is too high? Have you done a dilution series across the range? I find that as a range finder, 10 samples of a 1:10 dilution series of the lysate from neat is worth a try.

2)You don't mention whether you carry this out at room temp or in the coldroom. If you are doing this at room temp, then try mixing the DNA and protein in the coldroom and also running the gel at that temperature.

3)The fact that you have some complex being retained in the wells is a bit strange. Could you be precipitating your protein when complex formation occurs? Have a look at your binding buffer components and make sure that the pH and ionic components are ok? I don't know the make up of your binding buffer but I've included mine here for reference.

(25mM HEPES-KOH [pH7.9], 0.1mM EDTA, 5mM MgCl2, 50mM KCl, 1mM DTT, 20% (w/v) glycerol, 0.2mM PMSF, and 1ul protease inhibitor cocktail containing 1.6ug poly (dI-dC) . (dI-dC) (Sigma)

Hope this helps...

Keep posting...we'll get you through this...

Canalba, UK