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Possibly a stupid question.


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3 replies to this topic

#1 Doraid

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Posted 20 July 2017 - 04:26 PM

Greeting,

I was planning on running two gels. One with 50ug of protein per well and the other 20ug. I'm looking at a protein where I would need to load decent amount of the protein hence the 50ug.

The loading of the 20ug went smooth, as I was using the 10-well 1.5mm combs from Bio-Rad.

The 50ug not very well. My samples well at 63uL per well and those well take 66uL. 

Can I load half of my samples' volume, run the gel until the sample start entering the stacking gel and load the rest?

 



#2 mdfenko

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Posted 21 July 2017 - 07:12 AM

no. you will end up with two sets of bands (unless you are running ief, in which case you don't have a stacking gel).

 

you will just have to either concentrate your samples (prior to addition of sample buffer) to lower the volume necessary or carefully apply the sample so that it doesn't overflow the well.


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#3 labtastic

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Posted 21 July 2017 - 08:22 AM

I recommend acetone precipitate and resuspend dried pellet in 10-20ul 1x SDS sample buffer. 

 

https://tools.thermo...ecipitation.pdf

 

Generally speaking, I tend to see that smaller loading volumes give cleaner bands.

 

Plus you remove all the salts by precipitating. Excessive salts in your gel samples can cause some issues e.g. streaking depending on the salt and the quantity.


Edited by labtastic, 21 July 2017 - 09:01 AM.


#4 Doraid

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Posted 26 July 2017 - 04:59 PM

Sweet. Thank you for the help Labtastic and mdfenko.






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