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EMSA using biotin labeled probes and buffers


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#1 Mortis

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Posted 24 February 2010 - 05:55 PM

Hi All

Im new to EMSAs. Our lab has recently bought the Panomics EMSA kit (which uses biotin labeled-probe), probably much like the Pierce equivalent. As such it comes with all the binding buffers, wash buffers, block buffers etc.

I think it has worked, as in i get a bandshift in the control extract with the control labeled probe (and this disappears with the cold control probe). However, as we want to do lots and lots of shifts over next few months, we want to make our own buffers to save on costs (the kit is ~$600 in Australia!).

Ive made my own binding buffer ok (based on Hellman et al 2007), and i can make my labeled own probe easily enough, but what would people recommend to replace the block, wash and detection buffers. Im assuming BSA or skim milk power in PBS (or PBST) would be ok for a block buffer, but what of the wash buffer (just PBST?), and does this depend on the membrane i use? And what would the detection buffer be composed of? Can i use a western blotting ECL kit for detection of the membrane.

Answering these would make "doing it ourselves" a possibility

Thanks for any help

#2 aimikins

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Posted 24 February 2010 - 07:32 PM

I put together a kit several years ago when I was doing EMSAs. I posted the protocol up on this site:

http://www.protocol-...tocol-3455.html

I hope some of this can help you. it's tough to do it on the cheap!
"it is a miracle that curiosity survives formal education" -A.E.

#3 Mortis

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Posted 24 February 2010 - 09:48 PM

Hi aimikins, thanks for the reply

Yes ive had a look at that protocol before when researching this. Very handy, and thanks very much for posting. However i still have some questions:

1. There doesnt seem to be a blocking step in the NEB Phototope-Star kit manual as far as i can see. The kit instructions (which presumably start after crosslinking of the membrane has taken place) say to start with washing in Detection Solution A, then straight to Streptavidin and so on. Is this right?

2. Would your protocol depend of the type of membrane used? We have both the Pall Biodyne B and Genescreen Plus membranes (both nylon 0.45um positively charged). The NEB Phototope-Star kit recommends a neutral or slight positively charged membranes.

Thanks again

I put together a kit several years ago when I was doing EMSAs. I posted the protocol up on this site:

http://www.protocol-...tocol-3455.html

I hope some of this can help you. it's tough to do it on the cheap!



#4 aimikins

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Posted 25 February 2010 - 08:01 AM

hey -

no, there isn't a blocking step. I was very concerned about that myself, and actually contacted their tech support to make sure there wasn't some typo in the protocol. it worked fine anyways, and that's correct.

I used a genescreen membrane. you should be fine with that, too.
"it is a miracle that curiosity survives formal education" -A.E.

#5 Mortis

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Posted 25 February 2010 - 03:11 PM

Ok thanks again aimikins

Ive ordered the kit and will give it a try - fingers crossed!

hey -

no, there isn't a blocking step. I was very concerned about that myself, and actually contacted their tech support to make sure there wasn't some typo in the protocol. it worked fine anyways, and that's correct.

I used a genescreen membrane. you should be fine with that, too.



#6 Mortis

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Posted 20 May 2010 - 10:06 PM

Hi again

Recently ive tried doing non-radioactive EMSAs with the protocol you suggested aimikins, and it does give a much cleaner result with the agarose gel compared to polyacrylamide. I also use the NEB Phototope star detection kit and it seems to work well.

I see a single band, hopefully indicating a shift. However, one problem i have is that my cold probe does not compete away my observed shift. I thought i might just be seeing a non-specific band, however when i load a pure sample of the transcription factor protein (i.e. pure recombinant human Glucocorticoid Receptor) which should bind my probe (i.e. a well described Glucocorticoid Receptor binding sequence, otherwise known as a "GRE") instead of the nuclear extract i see the same thing - no competition from the cold probe. This has happened for every GRE probe ive tried. Oddly, the free probe band intensity does decrease noticeably in my competition lanes.

Ive made my probes both by buying biotin-labeled oligos (from Sigma) and also manually labeling oligos using the Pierce biotin-labeling kit. I add roughly equal molar amounts of sense and antisense together, mix, then use a PCR machine to heat to 95 degrees, then cool by 1 degree per cycle - for 70 cycles.

Obviously the cold probe is far more concentrated than the labeled probe, but is that possibly affecting the annealing so that i dont have a functional cold probe? Does anyone have any other ideas as to what is going on?

Thanks in advance

Ok thanks again aimikins

Ive ordered the kit and will give it a try - fingers crossed!

hey -

no, there isn't a blocking step. I was very concerned about that myself, and actually contacted their tech support to make sure there wasn't some typo in the protocol. it worked fine anyways, and that's correct.

I used a genescreen membrane. you should be fine with that, too.






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