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Protein extraction buffer preparation (please check for accuracy)


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6 replies to this topic

#1 chandch

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Posted 01 August 2013 - 03:06 AM

Dear all
I am preparing extraction buffer: 50mM Tris-HCl, pH 8.5, 5mM EDTA, 100mM KCl, 1% w/v dithiothreitol, 20% sucrose and protease inhibitor cocktail. I am just wondering, could you please check if I am doing it right way. I am preparing 100 ml extraction buffer

1) I prepared 50mM Tris-HCl , pH 8.5 (100 ml) 

2)and then I added all these chemicals to the above 100 ml extraction buffer: 5mM EDTA (0.146g), 100mM KCl (0.745g), 1% w/v dithiothreitol (1g), 20% sucrose (20g) and appropriate amount of inhibitor cocktail.

Is this the right way of doing extraction buffer? Please check

Thank you



#2 Podge

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Posted 01 August 2013 - 03:17 AM

Haven't checked the calculations for the EDTA and KCL, but it seems good. You can also make higher molar stock solutions for the KCL and EDTA for future use. Less weighing and you have less variation between the lysis buffers. You know all the stuff hanging on to the weighing boat etc...



#3 chandch

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Posted 01 August 2013 - 03:23 AM

Thank you. I am confused. In my calculation above I added directly  EDTA and KCL to 100 ml of 50mM Tris-HCl pH 8.5. How can I prepare high molar stock for EDTA and KCL

 

Thank you



#4 Podge

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Posted 01 August 2013 - 03:28 AM

for your EDTA you could make a 100x higher concetrated stock than you need. That would be 500mM. Then for your future lysis buffers you just need to add 1ml to 99ml of water to get that 5mM EDTA concentration. easy :)

 

Only thing is to look out for is how molar you can make those solutions (check solubility) and the stability. :)

 

But that is just a side note. If the buffer works and you only use that once in a while, don't bother with it. Never change a running system!



#5 chandch

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Posted 01 August 2013 - 03:54 AM

Thank you.



#6 mdfenko

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Posted 01 August 2013 - 05:08 AM

adding all of the components to the already diluted tris will further dilute your tris and your components will be more dilute than you expect.

 

podge is correct that you should prepare concentrated stocks of your components and add them to make your lysis medium. a concentrated tris stock should also be used so that you add all of your components then adjust to your final volume.

 

we normally prepare 1M stocks of tris buffer (at the required pH), 0.2->0.5M edta (adjusted to pH 7.4 or 8, edta will not dissolve until pH is above neutral). kcl stocks can be made up to 4M (it is soluble to ~4.1M), whatever is convenient.

 

just remember that the stocks and dry components combined should not bring you over 100ml. if under then you can adjust with water.


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#7 chandch

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Posted 01 August 2013 - 11:33 AM

Thank you






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