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SDS-PAGE High MW bands dissapear!


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#1 LabTryps

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Posted 17 February 2016 - 06:56 AM

Dear all,

 

Sorry if this has already been resolved previously. I could not find any solutions to our problem in past postings.

When we run SDS-PAGE gels (tris-glycine, home made, ca 8-12% T) our proteins start resolving OK. However, after a while, high molecular bands begin to dissapear. The problem can be as severe as to end up with no bands whatsoever at the end of the run. The gels are run at 70-120 Volts and never above 70 mA; also, they do not overheat, since they are cool to the touch when dissasembled.

I think we have tried changing every obvious item: reagents (tris, glycine, acrylamide...), water, even power supply and gel apparatus. It did not help.

The problem affects both samples and standards and it is not unlike the one explained here: http://www.protocol-...osts/42427.html.

I attach a picture with gel examples. One of them was run for a very short time on purpose to show that the separation starts OK but gets ruined as time goes.

 

Any help will be very wellcome! We are at our wits' end.

 

Cheers

Attached Thumbnails

  • Images gels no high MW bands.jpg


#2 bob1

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Posted 17 February 2016 - 11:18 PM

Some things that might be useful for us to know:

Recipes for your buffers and gels?

Amount of protein loaded?

How is it stained (pre-run? post-run?)

Dimensions of your gels?



#3 LabTryps

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Posted 19 February 2016 - 04:57 AM

Hi Thelymitra,

 

Our buffer system is the classical Laemli (we've also tried NuPage-like withouth success, though).

Amount of protein varies from 1-2 micrograms to 50 (we usually run alongside extracts, purifications, elutions and suchlike samples); it makes no difference.

Stains are all Coomasie Blue after gel run. The effects on the pre-stained MW markers are visible before staining, though.

Dimensions: minigels (6x8.5 cm) and acrylimide is usually around 10% T (ocasionally 8 and 12%). Changing acrylamide did not help.

 

Thanks in advance for your help



#4 LabTryps

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Posted 19 February 2016 - 05:24 AM

Sorry  Bob1. I got your name wrong!



#5 bob1

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Posted 19 February 2016 - 10:56 PM

Ok - those should be fine. Is there any indication that the pH of the gel is changing...are the prestained markers turning green or yellow?



#6 phage434

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Posted 20 February 2016 - 07:27 AM

The pH of the buffer would also be the first thing I would check. I would re-make the buffer and make sure it was at the correct pH and concentration.



#7 mdfenko

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Posted 22 February 2016 - 12:15 PM

another thing to check is the sds. if it's old then it may be decomposed enough to screw up the run.

 

try a newer lot of sds.

 

also, when you prepare the electrode buffer, do you adjust the pH? if you do then stop. the tris and glycine should be carefully weighed and brought to the proper volume. adjusting with naoh (or koh) and/or hcl will introduce unwanted salts to the solution.


Edited by mdfenko, 22 February 2016 - 12:16 PM.

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

#8 Andrea Fortina

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Posted 25 May 2016 - 06:49 PM

What is your protein size? Do you use a marker there, how about your marker? Do you use the same system to analysis protein had fine result before?


Andrea Fortina

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CreativeBiomart, Recombinant protein expert

 

 





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