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loading buffer and western blot help!


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#1 jasmina

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Posted 18 October 2010 - 01:25 PM

hi,
i would like to ask stupid question about the way you prepare the lammeli buffer. My way is the folowing, i have stock of loadinge dye 3x: (-20C). when i do sds-page gel, i just add my sample to loading dye, boil and load. on the other hand, i made sds page in another lab, and someone noticed that, im wrong, and i should add water to prepare my sample with loading dye! ex; if i want to load 50ug of protein from 10mg/ml of total extract and 32ul as final volume: i take 4.55ul, then, i add 10.6ul of loading buffer and then rest 16.85ul of water. this is correct but im not used to do that before!
what i do, is i take the volume of protein according to the concentration i want to load then, i add directly loading buffer 3x about 7ul, and i boil, and load!!
can this make difference in the result??
please, help, i know that it looks stupid, but, i need to understand, if i have to add water always??

#2 bob1

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Posted 18 October 2010 - 03:11 PM

You should always work such that your loading buffer is going to be at a 1x concentration when you load it onto the gel... i.e. if you have a 3x buffer, and 9ul of sample, you would add 3ul of loading buffer, mix, boil, etc., and load the whole lot onto the gel.

Edited to add: If the loading buffer is at the wrong concentration (too high or too low) when running, it can affect how your proteins migrate and/or the appearance of the lanes.

Edited by bob1, 18 October 2010 - 03:13 PM.


#3 mdfenko

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Posted 19 October 2010 - 11:50 AM

wouldn't that be 4.5ul 3x loading buffer for 9ul sample?
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#4 bob1

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Posted 19 October 2010 - 03:24 PM

Sorry, quite right, I was thinking 9 ul total volume.

#5 jasmina

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Posted 26 October 2010 - 06:23 AM

hi, thanks for your replies!
but, where is water in all this?
im wondering if adding water to fill until total volume before boiling is commun for you or not??
thanks

#6 mdfenko

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Posted 26 October 2010 - 07:17 AM

the way the dilution is presented here is without the use of water.

Edited by mdfenko, 26 October 2010 - 07:17 AM.

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