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Cup-loading: sample remains in cup

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Violeta G:
Hello everybody,

I'm a new user of the cup-loading technique and I'm having some problems with that. I'm trying to focus samples in the pH 6-11 range, so I am using passive rehydration followed by anodic cup-loading. So far, I've made two experiments but in both cases the sample remained in the cup at the end of the IEF protocol. I've read similar cases in the forum, but I have not been able to find a solution.

These are the details of the experiments:
Sample treated by CleanUp kit (GE) and finally resuspended in 7M urea, 2M thiourea, 4% CHAPS, 1,2% DeStreak, 1mM DTT, 0,5% IPGBuffer 6-11, bromophenol blue.
Strip: Immobiline DryStrip pH 6-11 (GE), 18cm.  
Passive rehydration of the strip: 16 hours, r/t, in 7M urea, 2M thiourea, 4% CHAPS, 1,2% DeStreak, bromophenol blue. First experiment without CoverFluid; 2nd, with 2ml aprox of CoverFluid.
Anodic cup-loading. Anodic paper wick: 100 ul of water; cathodic paper wick: 100ul of 7M urea, 2M thiourea, 4% CHAPS, 120mM DTT, bromophenol blue.

IEF protocol:
 First time:
30V-12h
500V-1h
grad 1000V-1h
grad 8000V-3h
8000V-6h

 Second time:
300V-3h
grad 1000V-6h
1000V-1h
grad 8000V-3h
8000V-4h

I've attached a photo of the IEF system just before the end of the protocol.

Any suggestions? Thank you all for you help.

Kjell:
Hi Violeta,

The problem is that the immobiline Drystrip is positioned in the wrong direction. The 6-11 gradient is labeled with a (-) at the cathodic end while the other strips are labeled at the anodic end with a (+). You should also remove the DeStreak from your sample but keep it in the DryStrip.

Good luck!
Kjell (GE)

Violeta G:
Hi Kjell,

thank you very much for your help. What a stupid mistake! I had just assumed that the orientation of the strips was the same in all of them. I'll try again following your suggestions.

SlavkoM:
Also, there will be some liquid remaining in the cup anyway. Don't worry about that, it's just buffer while proteins enter the strip and focus normally. The reason is that fully rehydrated strip just can't absorb all the liquid especially if the volume in the cup is larger.

In addition to Kjell:
Also remove DTT from anodic paper wick and replace it with 1.2% DeStreak. In any case, you should never have DTT and DeStreak in the same buffer. In the end you should have rehydration buffer (urea, thiourea, CHAPS, DeStreak, IPGBuffer) in the strip and in anodic paper wick, while sample in the cup contains urea, thiourea, CHAPS, DTT, IPGBuffer (or Pharmalytes).

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