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Protein labeling calculation - (Aug/04/2009 )

How to calculate this?

I need to label my protein. The dye should be 10 molar excess in reaction.
The labelled molecule need to be in 100 uM concentration.

Labelled molecule:
MW= 2000 (1mg/ml)
need to be diluted to 100 uM solution

M= 1 mg/ 2000 mg/mmol =5 x 10E-4 mM
V1=500ul = 500x 10E-6l
C1= 100 x 10E-6l

V2=c1 x V1/c2
V2= 100 ul
dilution: 100 ul molecule + 400 ul buffer


Dye:
MW= 720.66
C= 10mg (1mg/100ul DMSO)
M= 10mg/720.66 mg/mmol
0.013876 mM


10 molarity excess of dye to labelled molecule

M (of molecule)= 100 uM = 100 x 10E6 l
total volume of reaction= 20 ul = 20 x 10E6 l

Dye amount in reaction?
V2(dye)=c1 x V1/c2
V2(dye x 10 excess) =1.44 ul

is this right at all??

help highly appreciated!

-Joana-

I donít think so.
According to my calculations you have 13,876mM (not 0.013876 mM) dye and for 500ul reaction you should put 100ul molecule ( to have 100uM solution) and 36ul dye ( to have 1mM solution) and 346ul buffer.

Regards

-gotmog-

The total volume what I wanted for my labeling was 20 ul; I just wanted do a stock of 100 uM molecule first (ok unnnecessary I know ;).

So since you calculated that the tot. vol would be 500 (25 times more), so then my answer 1.4 is correct (for 20ul tot. volume). Right?



gotmog on Aug 4 2009, 09:21 AM said:

I donít think so. According to my calculations you have 13,876mM (not 0.013876 mM) dye and for 500ul reaction you should put 100ul molecule ( to have 100uM solution) and 36ul dye ( to have 1mM solution) and 346ul buffer.

Regards

-Joana-

And yes, it is 0.013876 M not mM (just wrote it wrong but calculated as M)!

Joana on Aug 4 2009, 09:54 PM said:

The total volume what I wanted for my labeling was 20 ul; I just wanted do a stock of 100 uM molecule first (ok unnnecessary I know ;).

So since you calculated that the tot. vol would be 500 (25 times more), so then my answer 1.4 is correct (for 20ul tot. volume). Right?



gotmog on Aug 4 2009, 09:21 AM said:

I donít think so. According to my calculations you have 13,876mM (not 0.013876 mM) dye and for 500ul reaction you should put 100ul molecule ( to have 100uM solution) and 36ul dye ( to have 1mM solution) and 346ul buffer.

Regards

-Joana-

Joana on Aug 4 2009, 11:27 PM said:

And yes, it is 0.013876 M not mM (just wrote it wrong but calculated as M)!

Joana on Aug 4 2009, 09:54 PM said:

The total volume what I wanted for my labeling was 20 ul; I just wanted do a stock of 100 uM molecule first (ok unnnecessary I know ;).

So since you calculated that the tot. vol would be 500 (25 times more), so then my answer 1.4 is correct (for 20ul tot. volume). Right?



gotmog on Aug 4 2009, 09:21 AM said:

I donít think so. According to my calculations you have 13,876mM (not 0.013876 mM) dye and for 500ul reaction you should put 100ul molecule ( to have 100uM solution) and 36ul dye ( to have 1mM solution) and 346ul buffer.

Regards




Yes, for 20 ul reaction 1.4ul dye is correct.

-gotmog-