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Lanes all run together


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#1 Louisiana_Sarah

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Posted 02 June 2010 - 10:58 AM

What is the reason for having all the lanes run together so it's just one big line across the gel? I am thinking something with the gel polymerization??? see pic

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#2 mdfenko

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Posted 04 June 2010 - 10:54 AM

i'm thinking about salts and how long after loading the gel it was started.

give more info about the gel and sample.

the running together looks like the bromphenol blue at the buffer front. could your label be travelling with the front? are you sure it was bound to your protein?

Edited by mdfenko, 04 June 2010 - 10:55 AM.

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#3 laurequillo

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Posted 04 June 2010 - 11:40 AM

What is the reason for having all the lanes run together so it's just one big line across the gel? I am thinking something with the gel polymerization??? see pic


I see that very often with Tubulin o actin. The same gel is ok if you check for protein X, but the tubulin is like that. I cannot tell you why...
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#4 Aris

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Posted 06 June 2010 - 12:29 PM

What is the reason for having all the lanes run together so it's just one big line across the gel? I am thinking something with the gel polymerization??? see pic



How long after loading do you wait before you start running the gel?
How many μg do you load?
I have also had this pattern before, but dont get crazy about it...
I think you have let the gel sit a while in the running buffer before you started running it.
Also I think you might have loaded quite a lot.

#5 Louisiana_Sarah

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Posted 07 June 2010 - 07:21 AM

More info:
Gel is 10% SDS-PAGE made according to the standard BioRad protocol
Samples are kidney membranes diluted 1:2 in Laemmli for a final concentration of 2.5 mg/ml
50 ug loaded per lane
Ran immediately after well loading at 120 V at RT

mdfenko: The dye front ran normally and was at the bottom of the gel at the end of running.

The BioRad Troubleshooting Guide says that "Lateral Band Spreading" is caused by:
1. Diffusion out of the wells prior to turning on the current
...but I turned on current immediately after loading)
2. Ionic strength of sample lower than that of gel
...these same exact samples ran perfectly on a precast gel, that is why I think it is something with the gel not the samples. i noticed that the leftover gel in the flask did not polymerize even though the cast gel did, that is why I was thinking maybe it was not COMPLETELY polymerized even though it was enough for me to layer the stacking gel.

Thanks for your help,
Sarah

#6 Aris

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Posted 08 June 2010 - 05:40 AM

It might be that it takes you a littel bit more to load all the samples. In that case the proteins from the first well start diffusing outside.
50 ug is a lot.
Try using a new fresh APS.
The APS has to be new and fresh each time you cast a gel. I have had issued before with that and it really makes a difference when the APS is new. If you have bought the APS quite some time ago try buying a new one and everytime that you are going to be casting a gel make the APS new and fresh.
In my experience with a new APS the gel polymerises in no more that 10 minutes. Furthermore try to use water saturated butanol in a mix
50:50 v:v to cover the resoving gel.
Let me know how it goes.

Cheers




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