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continual problems with 2d gel electrophoresis


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#1 biochem06

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Posted 05 January 2016 - 03:01 AM

Hi,

 

I would be very grateful if anyone had any input on what the problems are with my 2D gel. Gel A is human synovectomy tissue and Gel B is a cell line.The synovectomy sample was previously stored in RNAlater so I dialysed and did an acetone precipitation then rehydrated in standard 2d buffer. There is 200ug protein loaded on to an 11cm pH4-7 Bio rad strip. The IEF protocol is as follows:

1-step 500V 1hr.

2-gradient 1000V 1 hour.

3-gradient 6000V 2.5 hours

4- step 6000V 50 minutes.

 

Gel A is the worst.There are excessive amounts of vertical streaking. Also, there is a line across the gels at the bottom, even though the dye front was run right to the bottom of the gels .

 

Has anyone any ideas what could be causing the streaking and the line across the bottom?

 

many thanksgels.jpg



#2 mdfenko

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Posted 05 January 2016 - 05:36 AM

the sharp line is usually a buffer front (a component of the running buffer, usually the trailing ion). some tracking dyes run ahead of the front, some with and some behind. so, running the dye to the end of the gel will not necessarily eliminate this.

 

sometimes these effects are caused by decomposing (old) sds.

 

vertical streaks are often caused by incompletely solubilized proteins which solubilize during the run (in ief, the proteins migrate to the pH where they have no net charge and actually precipitate at that point. for the second dimension you resolubilize with sds buffer. this may not have been complete and leads to streaks up to the band or dot in 2d). they can also be caused by contamination in the system (often dust).

 

Are you casting any of the gels or are all of them commercial? if commercial,how near to or past the expiration date? if not, how old are the components?


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genius does what it must
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#3 biochem06

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Posted 05 January 2016 - 07:11 AM

Hi, thanks for your reply mdfenko. That's very useful information. The gels are commercial and are one year past expiration date. I know that's not ideal but I wanted to use them anyway while I'm trying to get this issue sorted with the samples. The SDS I'm not sure about, it's general lab stock but there is a chance it could be 2 or 3 years old.



#4 mdfenko

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Posted 06 January 2016 - 04:54 AM

i think if you rerun the samples with unexpired gels and fresh buffers (made from newer components, although 2-3 year old sds isn't awful) that you will attain publication quality results.

 

one more question, are you staining with silver or coomassie? silver is very good for showing slight aberrations in the gel.


talent does what it can
genius does what it must
i do what i get paid to do




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