Hi! I'm brand new to western blots and still learning how to calculate the concentration and how to reconstitute lyophilized proteins.
I have a 20ug vial I want to reconstitute, and we normally use 40ug/20ul for our western blot. So I believe 40ug/20ul = 2000ug/ml. So, if I reconstitute my 20ug vial to 10,000ug/ml, I would need to take out 4ul to have 40ug/20ul.
Is this correct? Any additional feedback would be greatly appreciated! Thank you.
Dear Alyssa,
your calculations are right, but you have to consider:
to get a concentration of 10,000 µg/ml with 20 µg total in your vial, you yould have to dilute it in 2 µl of solvent (10,000 µg/20 µg = 1:500 -> 1ml/500 = 2 µl). This means you cannot get your 4 µl.
I do not know if I get it right, but it looks to me that your vial does not contain enough protein to start with, when you want to load your Western Blot as usual with 40 µg in 20 µl.
All the best and stay healthy,
Marc
Marc Herb
Thank you for your response!
I used this online calculator https://www.rndsystems.com/resources/calculators to determine the amount I needed and it came out to 2000ul. Am I inputing the wrong values? Thank you for your help!
Marc Herb I entered the wrong value haha thank you so much for pointing that out!
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