Hi, hope all is well with you guys.
I have a question regarding the calculation of the volume of the protein sample prior to the loading for electrophoresis. I always make sure to load the same protein concentration in all wells (10 micrograms whenever possible). Now lets assume that that the total sample volume (protein+diluent+buffer) containing 10 micrograms of protein is 30 microliters. My question here is if I want to prepare a larger volume say 35 microliters, and only load 30 mciroliters in each well containing the 10 micrograms of protein, what calculation should I use. Many thanks.
Hi Moe,
If you would like to prepare 35 µl at a concentration of 10 µg/30 µl, you will need:
10 µg * 35/30 = 10 µg * 1.1666.... = 11,66... µg
And the same factor applies for the volume of sample buffer you would use to achieve the volume of 30 µl
Dear Moe
Your question is very good.
This is dependent to further analysis which you want to apply on the prepared PAGE:
1. If you want to evaluate and compare different proteins band by densitometry, you must run similar concentration in each well. therefore, you must calculate the concentration of proteins according to formula that presented by Christian as above.
2. if you want to compare that protein exist or lack in each samples, you need no similar concentration and can only use similar volume of loading.
Cheers
Your preferable protein concentration is 10ug/30ul which is 0.33ug/ul. For 35ul, the protein needed is 0.33*35 = 11.55ug, if 30ul from this preparation is loaded it will be 9.99ug (0.33*30 = 9.99)
I think it is the same with Christian Z's answer.
But for similar situations you could use this formula 10μg/30μl =xμg/35μl then you go cross multiply like that 10μg x 35μl=xμg x 30μg, then you solve the equation to get xμg=11.9μg. Hope it will help.
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