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Protein Extraction buffer (Please reply)

Posted: Thu Aug 01, 2013 2:53 am
by ssgrrens
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

Posted: Thu Aug 01, 2013 6:17 am
by Calvin8
You might consider making stock solutions as this is a better strategy for long term storage and convenience. I suggest making the following stock solutions and then diluting appropriately:

1M Tris-HCL pH 8.5
500mM EDTA
1M DTT (store aliquots at -20C)
300 mM Sucrose

good luck

Posted: Thu Aug 01, 2013 11:29 am
by ssgrrens
Thank you. I understand. But how can I convert molarity to percentage for DTT and Sucrose? I need 1% DTT and 20% sucrose?