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can anyone help me with calculating a molar ratio of protein to lipid?


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#1 loz

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Posted 08 August 2013 - 10:16 PM

Hi,

 

Can anyone help me with a calculation?

 

I have expressed and purified my protein(MW33.57) via His tag and gel flitration  and have calculated the molar concentration using the absorbance (1mg/ml) and mass extinction coefficient (.802) giving me a molar concentration of 37mM.

 

My lipid (FW 677.93, 33.89mg) was reconstituted, dried and solubilised to 1ml with a 50mM concentration.

 

I would like to know how to calculate a 1:160 (protein: lipid) molar ratio to combine the two so that I can assemble these together via removal of detergent, then a gel filtration step Superdex 200 column, 100ul to 300ul injection volume.

 

Cheers

loz



#2 Trof

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Posted 14 August 2013 - 03:53 AM

Your relative molar mass values (MW and FW) are strange. Single aminoacid has about 110 Daltons, yet you write your "protein" has 33.57.

 

Also I didn't calculate the rest, but I don't get what you ask exactly, when it's 1 mole of protein to 160 moles of lipid and you said to have the molar concentrations of both.


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#3 loz

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Posted 17 August 2013 - 11:54 PM

Thanks Trof

 

Hmmmm…your reply is another sign that I have no idea what I am doing….I’m new to dealing with proteins. If you can persevere, let me tell some back ground and how I arrived at things.

 

I am trying to put together an empty nanodisc which has two components, an MSP scaffold protein which encircles a phospholipid, DMPC. The idea is that you bung them together at a particular ratio along with biobeads, remove the detergents, once removed they form a disc. In that disc you can assemble trans-membrane proteins and hope to make them soluble. But for now I want to see if I can make an empty one.

 

Firstly, the MSP protein was purified via His and gel filtration to clean it up. I fed the sequence into ExPASy ProtParam which predicted my mass extinction co-efficient at 0.802. My A280 absorbance (nanodrop) readout was 1mg/ml.

I calculated absorbance/extinction co=efficient  ie. 1.0 / 0.802=1.25mg/ml (g/L).

Then mg.ml/MW (g/mol)=Da, =M concentration? ie. 1.25mg.ml/33.57g.mol=37mM MSP protein?????

I might have done this wrong or used wrong terminology……

 

The second component is DMPC which came as a powder. On the jar is was labeled 33.89mg, FW677.93. I reconstituted 50mM using chloroform (33.89mg in 1 ml), dried it out with N and freeze dry, then re-suspended it in 100mM Sodium cholate (volume to volume).

 

My next step is to put them together incubating with biobeads, but the ratio must be 1:160 (MSP:DMPC) for it all to fit, then gel filtration max volume of 300ul. The protocol (Ritchie et al., (2009), Sligar lab) did not specify what type of ratio it was, I assumed molar.

 

Cheers!

 

Loz



#4 Trof

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Posted 18 August 2013 - 02:04 PM

I never did anything like this but there are several strange things anyway.

 

What I got that you are trying to calculate molar concentration from mass concentration, which you try to calculate from absorbance.

I don't remeber those equation right away and it's way too late, but:

 

For example this MSP protein has relative molar mass over 24 000 Da and extinction coeficient  21 000 M-1cm-1

I don't know what kind of scaffold protein you have, but these values are just waaaay far from yours.

 

Also, you constantly give those weird extinction coefficient values in unitless format (0.802) and absorbance values (which are unitless) in 1mg/ml. So god knows what are you calculating.

Nanodrop as I remember gives already mass concentration, at least for DNA/RNA, maybe it does the same for proteins if you fill in the right values first.

 

So please check out what are you getting from ExPASy and what are you actually getting from Nanodrop.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#5 loz

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Posted 19 August 2013 - 02:48 AM

Thanks Trof, I am very grateful for your attention to my problem. If i ever work it out I will give you a quick line to let you know. I think I will "suck it and see".

 

Thanks!!!!






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