Author Topic: Destreaker kit  (Read 8497 times)

bokajsen

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Destreaker kit
« on: April 20, 2009, 05:00:00 PM »
I am currently optimizing a DIGE experiment. I plan to use pI 3-10NL strips because the sample contains many basic proteins, however, I see some streaking in the range above pI 7 (as can be expected). So I have now bought the Destreaker kit.
Questions:
1) Is it true that the destreaker kit only works with cup-loading (which I do not plan to use)?
2) How do I use it? Sorry, but the Destreaker protocol is a bit confusing. Do I mix the destreaker solution with the IPG buffer and add sample plus DTT (final conc:1mM) and then load it into the "strip-holder" and run as normal?


Sjouke Hoving

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Re: Destreaker kit (bokajsen)
« Reply #1 on: April 21, 2009, 05:00:00 PM »
The protocol is very easy: yes, you have to use cup-loading with DeStreak. You rehydrate the strip with buffer + DeStreak only. You apply the sample (ussually containing some DTT) by cuploading. You should never mix DTT and DeStreak in the strip, because then DeStreak becomes Streak... formation of B-mercaptoethanol you want to avoid in IEF. I guess, when you don't use DTT at all, you might try in-gel rehydration as well (but I never have tried this).

sjouke


Kjell

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Re: Destreaker kit (sjouke)
« Reply #2 on: April 21, 2009, 05:00:00 PM »
You are right that the instructions for the DeStreak Reagent says that no reducing agents can be used in rehydration loading. The general advice is to not mix DTT and DeStreak.
However, if your sample contains DTT you can still use it in rehydration loading with DeStreak, provided that the final concentration in the rehydration solution does not exceed 1mM (see DeStreak Rehydration Solution). Never add DTT directly to a rehydration solution with DeStreak, though.

Kjell

Kjell (GE Healthcare)

Bcrespo

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Re: Destreaker kit (bokajsen)
« Reply #3 on: April 21, 2009, 05:00:00 PM »
Hi have used Destreak with rehydration in gel, and it works great.

The lysis buffer contains only 10mM of DTT (not 65mM as usual). And of course, don't add DTT to the Rehydration buffer.  

Good luck

Bcrespo

Bcrespo

bokajsen

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Re: Destreaker kit (Bcrespo)
« Reply #4 on: April 21, 2009, 05:00:00 PM »
Thanks for the tips.
I forgot to mention: my sample is in Urea/Thio-urea/CHAPS/Tris buffer, but without! DTT.
So here is simply what I will do:
Mix 9 ul IPG buffer (pI 3-10) (for 2% solution), 431 ul Destreak solution and 10 ul Sample (no DTT!) and load on 24cm 3-10 strips by rehydration (no cup-loading) as normal, right?

dfried

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Re: Destreaker kit (bokajsen)
« Reply #5 on: April 21, 2009, 05:00:00 PM »
You may find that anodic cuploading will give you the best results in this case, but as you can read, others seem to do just fine via rehydration loading.  You might want to try both just to compare for yourself.

With anodic cuploading on pH 7-11 24 cm strips rehydrated in DeStreak rehydration solution plus 0.5% IPG buffer, we have about 13 mM DTT in the sample (tolerance specs are 20 mM, I think), which works very well in most cases.

David


DrHollyday

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Re: Destreaker kit (dfried)
« Reply #6 on: April 21, 2009, 05:00:00 PM »
rehydration loading with bis-mercaptoethanol works fine.

Sjouke Hoving

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Re: Destreaker kit (DrHollyday)
« Reply #7 on: April 22, 2009, 05:00:00 PM »
I am curious to see your gel. Can you show an image of a gel using mercapto ethanol?

sjouke


DrHollyday

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Re: Destreaker kit (sjouke)
« Reply #8 on: April 22, 2009, 05:00:00 PM »
bis-mercaptothanol is destreak

bokajsen

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Re: Destreaker kit (DrHollyday)
« Reply #9 on: April 22, 2009, 05:00:00 PM »
Sure I will provide you with a picture (unless its a complete disaster  )

ElkeKS

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Re: Destreaker kit (DrHollyday)
« Reply #10 on: April 22, 2009, 05:00:00 PM »
No, DeStreak is hydroxyethyldisulfide (HED). As Sjouke points out, it will react to 2-mercaptoethanol when mixed with DTT. And I believe he would like to see an image of a gel run with 2-ME, because it is not a recommended reductant as it would cause streaking in the basic range.
Elke

DrHollyday

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Re: Destreaker kit (ElkeKS)
« Reply #11 on: April 23, 2009, 05:00:00 PM »
As mentioned before I do not used 2-ME. I used bis- (not ß) 2ME.

I thougth it is an usualy name for 2,2'-dithioetanol.

Like Bis-tris.

http://en.wikipedia.org/wiki/Bis-tris" TARGET="_blank">http://en.wikipedia.org/wiki/Bis-tris


Sjouke Hoving

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Re: Destreaker kit (DrHollyday)
« Reply #12 on: April 23, 2009, 05:00:00 PM »
Thanks ElkeS.... yes I would like to see a gel with your Bis-mercapto...

sjouke


DrHollyday

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Re: Destreaker kit (sjouke)
« Reply #13 on: April 29, 2009, 05:00:00 PM »
good to show a gel,

This is a seperation of a lipd raft fraction run with bis-mercaptothanol

best wishes


Bcrespo

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Re: Destreaker kit (DrHollyday)
« Reply #14 on: April 29, 2009, 05:00:00 PM »
You have good separation in acid and neutral part, but your basic part is lost!! Maybe you can improve it using REAL DESTREAK reagent!!  

Best,

Jose

Bcrespo