Protein Purification Calculations

[RandomFactor]

Hi,

 

This is my first post, I hope of many more to come, but at the moment I'm in need of a bit of help...

 

Basically, I have purified a protein and I wanted to know its yield, but I'm not sure I'm doing the calculations right (its not an enzyme)...

 

Here goes:

 

The initial volume of the Flask in which I made the protein was 2 Litres.

The final volume of the cuvette was 600 uL (microlitres).

 

Concentration of Protein in cuvette was (there were 3 samples):

 

1) 591 uM (micromolar)

2) 456 uM

3) 2141.8 uM

 

The molecular wieght of the protein was 7396.2 (MW).

 

Anyone got any idea about how to go about calculating the yield?

 

I worked out the molar quantity in the cuvette (vol x conc) and then multiplied it by 7396.6 to get the wieght of the protein (in milligrammes). I then used this figure and divided it by 2 Litres which I had in the beginning, but I'm getting some really strange figures (98%'s).

 

Anyone know where I went wrong?

 

Thanks very much.

 

[bTW. I kind of need this for a write up I have to hand in on Monday, so any quick help would be REALLY appreciated lol)

[donkey]

hi there,

 

can you explain exactly how you went from 2 Litres to 3x600 uL? And what did you know about the initial 2L sample? Did all 3 600uL come from the same 2L stock or did you have 3x 2L initial samples?

 

In general, my understanding of a yeild is that it's the amount you got at the end relative to the maximum possible amount (in a purification procedure the max possible amount may be the same as the starting mass of the protein you're interested in. This yeild can be expressed as a percentage.

 

For example, lets say you had 2mg of your protein in 2L at the start (but this was an impure sample - your total protein concentration/mass including all the other proteins was way above 2mg). If after purification you had 600uL containing only 1 type of protein (i.e. a pure sample) you can now work out the yeild from the concentration/mass of protein you have.

 

So if you found out that you had 2mg in your final 600uL sample then your yield you be 100%.

If you had 1mg in 600uL, your yield would be 50%.

 

The hard part is knowing what your initial specific (i.e. the protein you're interested in) protein mass / concentration was compared to your final sample.

 

If you used an enzyme and assayed for enzyme activity at the start and end you could work it out. Do you have any way of knowing the concentration/amount of your protein you had in the first 2L sample (not total protein concentration but just the concentration or amount of the protein you ended up with).

 

I hope some of that helps and please post back if you need some more help to clarify things

[RandomFactor]

Hi

 

Thanks for your reply.

 

I had a cell culture, which contained an unknown amount of protein, and then i passed them through a "french press", basically bursting the cells open, seperated the gunk from the proteins. Then I used a TALON column (His-Tag) for the first step of purification, and then I used a gel filtration colomn for the second step.

 

I then used a concentrator to concentrate the protein into a 600uL sample (for NMR studies).

 

So I don't really know what the original concentration of my protein was at all, and am finding it a devil of a time trying to figure out what the yield should be.

 

Any ideas lol?

[donkey]

sorry, you've got me stumped!

 

hopefully someone else here will have an idea although I appreciate you're running out of time

 

good luck!

[Bluenoise]

Hmmm I think you need to be more clear. I'm having trouble understanding what you're asking exactley. Yeild of what from what?

Yeild is how much you get.

% yeild is how much you get devided by some total, be it total protein content, total cell weight, expected yeild of the protein, yeild before purification, etc...

I you don't know what this total is then you can't calculated the %yeild. Simple as that.

Now you haven't said if the yeild is of a particular protein out of all the proteins, which sounds very likely since you seem to be purifying the proteins by gel exclusion. So then you must have recorded the total protein before putting them through the column or your out of luck.

You mention 3 cavettes, two with simular values one very different. What did you do to get these?

I'm sorry it's probably too late to help now anyways.

 

Where the confusion really lies is that you're talking about a percentage. But you haven't mentioned what the total of the percentage is, which makes it a very arbitrary figure, nor have you given any data to suggest how to calculate anything but final mass/moles of protein.

I wish I could help.

[RandomFactor]

I got a day's extension

 

Thanks guys, I asked my supervisor and he told me I'm not working out % yield, but just a yield. Basically that means I should be working out mg/L not a percentage.

 

The answers I got were correct, but they were in units of mg/L, and not % yield.

 

Sorry about the red herring , and thanks ever so much for the help.

[donkey]

yay, glad to hear you got it sorted

 

sorry, I'm more used to yields in % and didn't even think about anything else.