User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/06/25/Liposomes and kinesin: Difference between revisions

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The next step ideally is to put the liposomes under vacuum for a few hours to remove the rest of the chloroform. Right now I'm struggling to setup the aspirator to do this because we do not have a vacuum pump.
The next step ideally is to put the liposomes under vacuum for a few hours to remove the rest of the chloroform. Right now I'm struggling to setup the aspirator to do this because we do not have a vacuum pump.
And...it can't be done. So I'm not going to worry about it. I will keep blowing N<sub>2</sub> over it for an extended period of time though.

Revision as of 14:27, 25 June 2009

Calculating the amount of liposomes needed

The first thing I want to do is to calculate the amount of liposomes we will be using to coat each chamber. To do this I will start with the surface area for a lipid from a paper that I will need to find the reference for.

[math]\displaystyle{ 0.4 \mathrm{\ } \frac{\mathrm{nm}^2}{\mathrm{Lipid}} }[/math]

I have 100 nm filters that will make (nominally) 100 nm diameter liposomes. The number of lipid molecules per liposomes is

[math]\displaystyle{ \begin{align} \mathrm{Surface\ area\ of\ a\ liposome} = 4\pi r^2&\approx 31416\mathrm{\ nm}^2\\ \mathrm{Surface\ area\ of\ the\ bilayer\ of\ a\ liposome} = 2\times 4\pi r^2 &= 62832\mathrm{\ nm}^2\\ \frac{\mathrm{Lipid}}{\mathrm{Liposome}} = \frac{62832}{0.4} &= 157080\mathrm{\ }\frac{\mathrm{Lipids}} {\mathrm{Liposome}} \end{align} }[/math]

Using the average molecular weight of L-α-phosphatidylcholine of 770 g/mole, I can find the number of liposomes I can make with the 25 mg of lipid molecules I have.

[math]\displaystyle{ \begin{align} \left(770\mathrm{\ }\frac{\mathrm{g}}{\mathrm{mole}}\right)^{-1}\mathrm{\ }6.022\times10^{23}\mathrm{\ }\frac{\mathrm{Lipids}}{\mathrm{mole}} &= 7.8\times 10^{20}\mathrm{\ }\frac{\mathrm{Lipids}}{\mathrm{g}}\\ 7.8\times 10^{20}\mathrm{\ }\frac{\mathrm{Lipids}}{\mathrm{g}}\mathrm{\ }25\times 10^{-3}\mathrm{g} &= 2\times10^{19}\mathrm{\ Lipids}\\ \mathrm{\#\ of\ Liposomes} &= 1.2\times10^{14}\mathrm{\ Liposomes} \end{align} }[/math]

The next thing I want to find is the amount of liposome solution required to fully coat the surface area of our chamber. We are using cover slips and cover glass slides that are 24 mm and 25 mm in width respectfully.

[math]\displaystyle{ \begin{align} \mathrm{Surface\ area\ of\ chamber} &= 600\times10^{12}\mathrm{\ nm}^2\\ 1.2\times 10^{14}\mathrm{\ Liposomes} / 2.5 \mathrm{\ mL} &= 48\times10^{12}\mathrm{\ }\frac{\mathrm{Liposomes}}{\mathrm{mL}}\\ 600\times 10^{12}\mathrm{\ }\frac{\mathrm{nm}^2}{\mathrm{Sample\ chamber}}\mathrm{\ }\frac{\mathrm{Liposome}}{31416\mathrm{nm}^2} &= 1.9\times 10^{10}\mathrm{\ }\frac{\mathrm{Liposomes}}{\mathrm{Chamber}}\\ \frac{1.9\times 10^9\mathrm{\ }\frac{\mathrm{Liposomes}}{\mathrm{Chamber}}}{48\times 10^{12}\mathrm{\ }\frac{\mathrm{Liposomes}}{\mathrm{mL}}} &= 0.4\mathrm{\ }\frac{\mathrm{mL}}{\mathrm{Chamber}} \end{align} }[/math]

Thus the amount of liposome sauce needed to coat our sample chambers is

[math]\displaystyle{ \begin{align} 400\mu\mathrm{L} \end{align} }[/math]

Making liposomes

The lipid molecules come packed in chloroform. We need to get rid of this chloroform by using my patented SpinMaster Micro™.

This device takes the place of a much more expensive rotary evaporator. The idea is to blow nitrogen into the vial that has the lipid molecules and spin it. This evaporates the chloroform and leaves a nice uniform coating of lipid molecules in the vial. From experience, it is much easier to get lipid molecules off of glass when they are in a uniform film as opposed to clumps. Clumps happen when you try to evaporate the chloroform by hand rotating the vial.

The next step ideally is to put the liposomes under vacuum for a few hours to remove the rest of the chloroform. Right now I'm struggling to setup the aspirator to do this because we do not have a vacuum pump.

And...it can't be done. So I'm not going to worry about it. I will keep blowing N2 over it for an extended period of time though.